N-acylethanolamide derivatives and uses thereof

ABSTRACT

The present disclosure provides certain N-Acylethanolamide derivatives, and uses relating thereto.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/349,548, which is a U.S. national phase entry under Section 371 ofInternational Application No. PCT/US2017/056353, filed on Oct. 12, 2017,which published as WO/2018/071679 on Apr. 19, 2018, which claimspriority to U.S. provisional application No. 62/517,344, filed Jun. 9,2017, and U.S. provisional application No. 62/407,796, filed Oct. 13,2016. The entire contents of each of the prior applications are herebyincorporated by reference herein.

BACKGROUND

N-acylethanolamides are uniquely useful and valuable compounds. Studieshave shown that N-acylethanolamides can be effective in the treatment ofa variety of diseases, disorders, and conditions.

SUMMARY

N-acylethanolamides are widely recognized as potentially usefultherapeutic compounds, and have been extensively studied in particularfor their analgesic and/or anti-inflammatory effects.

However, the present disclosure appreciates that N-acylethanolamidecompounds often suffer from one or more poor pharmacological properties,for example resulting in limited bioavailability when administered by aparticular route (e.g., oral) and/or low exposure to a particular targetsite of interest (e.g., bowel or, more specifically, lower bowel). Inmany cases, these poor properties can limit dosage, limit exposure ordelivery to a particular site of interest, limit susceptibility toeffective delivery by a particular route, etc and/or may therefore orotherwise necessitate alternative modes of administration.

The present disclosure further appreciates that some or all of the poorpharmacological properties encountered with many N-acylethanolamidecompounds may be relieved or obviated by provision of an appropriateprodrug. As is known in the art, a prodrug is in most cases apharmacologically inactive derivative of a parent drug molecule thatrequires spontaneous or enzymatic transformation within the body inorder to release the active drug. The present disclosure encompasses theinsight that one source of pharmacological problems with certainN-acylethanolamide compounds may be failure of the compound to reach arelevant target site in sufficient level to achieve its desiredbiological effect. Alternatively or additionally, an N-acylethanolamidecompound may interact with non-target sites, and/or may displayundesirable side effects. The present disclosure provides certainderivative (e.g., prodrug) forms of N-acylethanolamide compounds thatmay relieve or obviate such problem(s) and/or source(s).

In some embodiments, the present disclosure provides derivatives ofN-acylethanolamides that exhibit improved pharmacological propertiesand/or display biological activity that is reasonably comparable to (or,in some cases may be better than) that of its parent N-acylethanolamide(or another appropriate reference N-acylethanolamide). In someembodiments, a provided N-acylethanolamide derivative compound mayexhibit one or more properties such as, for example, increased oralbioavailability, increased cell permeability, increased watersolubility, reduced first-pass effect, increased stability, activetransport by intestinal transporters, avoidance of efflux transporters,and/or combinations thereof when compared to a referenceN-acylethanolamide such as, for example, its parent N-acylethanolamide.

In some embodiments, a compound for use in accordance with the presentdisclosure is one wherein an N-acylethanolamide is conjugated to amoiety selected from the group consisting of phosphate, butyric acid,glycerol, succinate, caprylic acid, gluconoic acid, eicosapentaeonoicacid, linoleic acid, succinate, and sucrose moieties, and combinationsthereof. In some embodiments, an N-acylethanolamide is conjugated to oneor more such moieties through use of a linker moiety. In someembodiments, an N-acylethanolamide is conjugated to two or more suchmoieties. In some embodiments, an N-acylethanolamide is conjugated toone, two, or three such moieties.

In some embodiments, a provided compound has a chemical structurerepresented by formula I-a:

X₁-X2

-   -   or a pharmaceutically acceptable salt thereof; wherein    -   X₁ is an N-acylethanolamide; and    -   X₂ is a moiety conjugated to the N-acylethanolamide.

In some embodiments, X₁ is selected from the group consisting ofN-palmitoylethanolamide, N-oleoylethanolamide, andN-arachidonoylethanolamide; in some particular embodiments, X₁ isN-palmitoylethanolamide. In some embodiments, X₂ comprises a moietyselected from the group consisting of phosphate, butyric acid, glycerol,succinate, caprylic acid, gluconoic acid, eicosapentaeonoic acid,linoleic acid, succinate, and sucrose moieties.

In some embodiments, a provided compound has a chemical structurerepresented by formula I:

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   each R¹, R², or R³ is independently hydrogen or -T-R⁴, wherein        at least one of R¹, R², or R³ is -T-R⁴;    -   -T- represents a bivalent moiety; and    -   R⁴ is an optionally substituted group selected from the group        consisting of C₁₋₄₀ aliphatic, —C(O)R, and X₁: wherein        -   R is selected from the group consisting of hydrogen and            optionally substituted C₁₋₂₀ aliphatic; and        -   X₁ is as defined above.

In some embodiments, a provided compound has a chemical structurerepresented by formula I-b:

X₁-X₃   I-b

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   X₁ is as defined above;    -   X₃ is an optionally substituted group selected from the group        consisting of —(CH₂)_(m)—P(O)(OR)₂, C₁₋₄₀ aliphatic -T-X₄;        further wherein        -   m is an integer select from the group consisting of 0-10;        -   -T- is as defined above;        -   X₄ is a saccharide moiety, in some particular embodiments,            X₄ is a disaccharide, for example, sucrose.

In some embodiments, the present disclosure provides compounds such as,for example:

In some embodiments, the present disclosure provides N-acylethanolamideprodrugs. In some embodiments, a provided prodrug may be characterizedby one or more desirable physical properties, which may, for example, beassessed relative to an appropriate reference N-acylethanolamide (e.g.,to the parent N-acylethanolamide of the provided prodrug); in someembodiments, such desirable physical properties may be or include, forexample, enhanced aqueous solubility (which may facilitate, for example,formulation into a pharmaceutical composition, particularly for oral orparenteral administration), enhanced absorption from the digestivetract, enhanced stability under relevant storage conditions, etc.

In some embodiments, a parent N-acylethanolamide compound is one that ischaracterized by limited aqueous solubility and/or limited oralbioavailability. For example, in some embodiments, a parentN-acylethanolamide compound is characterized by aqueous solubility belowa relevant threshold and/or oral bioavailability below a relevantthreshold.

In some embodiments, a parent N-acylethanolamide compound ischaracterized by one or more pharmacological properties that impact itsamenability to pharmaceutical formulation, for example, so thatchallenges are encountered preparing pharmaceutical compositionscontaining a desirable unit dose amount and/or a desirable concentrationof the compound. In some embodiments, the present disclosure providesderivatives of such parent N-acylethanolamide compounds; in someembodiments, derivatives provided by the present disclosure act asprodrugs of the relevant parent compounds in that, when administered toa subject (e.g., in the context of a pharmaceutical composition), theprovided derivatives deliver the parent compound and/or an activemetabolite thereof. In some embodiments, as described herein, providedN-acylethanolamide derivative compounds comprise one or more moietiesmodifying or otherwise linked to a parent N-acylethanolamide compound.

In some embodiments, provided N-acylethanolainide derivative compoundsare amenable to pharmaceutical formulations at unit doses and/orconcentrations that are higher than those achieved with the relevantparent N-acylethanolamide compounds under comparable conditions.

Among other things, the present disclosure provides compounds of formulaI and pharmaceutically acceptable salts thereof, the preparation of theabove-mentioned compounds, medicaments containing them and theirmanufacture, as well as technologies for identifying and/orcharacterizing useful such compounds, and use of provided compounds, forexample in the treatment of one or more diseases, disorders, orconditions, for example as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart indicating pain classification and representativeindications. Distinguishing between different types of pain is criticalfor proper treatment. Pain can be classified by its duration into acuteand chronic pain. Chronic pain is further classified by the source ofpain production: nociceptive pain, which is transmitted by nociceptorsfrom the site of injury or tissue damage (for example, inflamed jointsin arthritis); neuropathic pain, which is initiated or caused by aprimary lesion or dysfunction in the nervous system (further subdividedinto central and peripheral, involving the central and peripheralnervous systems, respectively); visceral pain, which involves theinternal organs; mixed pain, which is of mixed origin. Prevalence forselected chronic pain conditions in the United States is indicates.Sources: Centers for Disease Control and Prevention, National Center forHealth Statistics, Arthritis Foundation, National Institutes of Diabetesand Digestive Kidney Diseases, American Pain Society, The American PainFoundation.

FIG. 2A is a bar graph illustrating visual analog 10-point scalemeasuring use of PEA to manage pain.

FIG. 2B is a bar graph illustrating quality of life over a 24-pointscale for patients administered PEA to manage pain.

FIG. 2C is a scatter plot measuring changes in pain intensity inpatients treated with PEA and control groups at different observationtimes. Values are expressed as mean±SEM.

FIG. 2D is a bar graph measuring a visual analog of pelvic pain amongGroups A, B, and C.

FIG. 2E is a bar graph measuring a visual analog of dyspareunia amongGroups A, B, and C.

FIG. 2F is a bar graph measuring a visual analog of dysmenorrhea amongGroups A, B. and C.

FIG. 2G is a bar graph illustrating percentage regularization of statusfollowing treatment with butyric acid and insulin. In particular, 15IBS-DP patients vs. 4 IBS-CP patients: 68% vs 14% and 71% vs 16%respectively in the intent to treat (ITT) and per-protocol (PP) groups(p<0.005). *Statistically significant (p<0.005).

FIG. 2H is a scatter plot illustrating the endoscopic score (n=10) inpatients with distal UC before and after treatment with sodium butyrate(black dot) or sodium chloride (white dot; control) enemas. Verticalbars indicate 1 SEM; *significant differences (endoscopic score, P<0.01;histological grading, P<0.02; upper-crypt labeling, P<0.03; Wilcoxontest).

FIG. 2I is a scatter plot illustrating the histological grading (n=10)in patients with distal UC before and after treatment with sodiumbutyrate (black dot) or sodium chloride (white dot; control) enemas.Vertical bars indicate 1 SEM; *significant differences (endoscopicscore, P<0.01; histological grading, P<0.02; upper-crypt labeling,P<0.03; Wilcoxon test).

FIG. 2J is a scatter plot illustrating the upper-crypt labelingfrequency (n=6) in patients with distal UC before and after treatmentwith sodium butyrate (black dot) or sodium chloride (white dot; control)enemas. Vertical bars indicate 1 SEM; *significant differences(endoscopic score, P<0.01; histological grading, P<0.02; upper-cryptlabeling, P<0.03; Wilcoxon test).

FIG. 2K is a bar graph illustrating inhibitory effect of PEA (1-10mg·kg⁻¹, i.p.) on upper gastrointestinal transit in control mice.Transit was measured 28 days after OM or vehicle (30% ethanol)administration. Results (the means±SEM of 9-10 mice for eachexperimental group) are expressed as a percentage of uppergastrointestinal transit. *P<0.05, **P<0.01, significantly differentfrom vehicle. The term “vehicle” refers to the vehicle used to dissolvePEA.

FIG. 2L is a bar graph illustrating inhibitory effect of PEA (1-10mg·kg⁻¹, i.p.) on upper gastrointestinal transit in mice treated with OM(oil of mustard). Transit was measured 28 days after OM or vehicle (30%ethanol) administration. Results (the means±SEM of 9-10 mice for eachexperimental group) are expressed as a percentage of uppergastrointestinal transit. *P<0.05, **P<0.01, significantly differentfrom vehicle. Note that in (B) the term “vehicle” refers to the vehicleused to dissolve OM. the % transit of a vehicle or PEA in OM-treatedmice.

FIG. 2M is a bar graph illustrating 2,4,6-dinitrobenzenesulfonicacid-induced (“DNBS-induced”) colitis in mice. Changes in body weightfrom control and DNBS-treated mice in the presence or absence ofintraperitoneal (i.p.) PEA. Mice were weighed before DNBS (or vehicle)administration and immediately before killing. Tissues were analysed 3days after vehicle or DNBS administration. PEA (0.1-10 mg·kg⁻¹) wasadministered once a day for three consecutive days starting 24 h afterthe inflammatory insult (therapeutic protocol). Bars are mean±SEM of12-15 mice for each experimental group. #P<0.001 versus control (i.e.mice without intestinal inflammation). *P<0.05, **P<0.01 and ***P<0.001versus DNBS alone.

FIG. 2N is a bar graph illustrating DNBS-induced colitis in mice.Changes in body weight from control and DNBS-treated mice in thepresence or absence of orally administered (p.o.) PEA. Mice were weighedbefore DNBS (or vehicle) administration and immediately before killing.Tissues were analysed 3 days after vehicle or DNBS administration. PEA(0.1-10 mg·kg⁻¹) was administered once a day for three consecutive daysstarting 24 h after the inflammatory insult (therapeutic protocol). Barsare mean±SEM of 12-15 mice for each experimental group. #P<0.001 versuscontrol (i.e. mice without intestinal inflammation). *P<0.05, **P<0.01and ***P<0.001 versus DNBS alone.

FIG. 2O is a bar graph illustrating DNBS-induced colitis in mice.Changes in colon weight/colon length ratio from control and DNBS-treatedmice in the presence or absence of i.p. PEA. Mice were weighed beforeDNBS (or vehicle) administration and immediately before killing. Tissueswere analysed 3 days after vehicle or DNBS administration. PEA (0.1-10mg·kg⁻¹) was administered once a day for three consecutive days starting24 h after the inflammatory insult (therapeutic protocol). Bars aremean±SEM of 12-15 mice for each experimental group. #P<0.001 versuscontrol (i.e. mice without intestinal inflammation). *P<0.05, **P<0.01and ***P<0.001 versus DNBS alone.

FIG. 2P is a bar graph illustrating DNBS-induced colitis in mice.Changes in colon weight/colon length ratio from control and DNBS-treatedmice in the presence or absence of p.o. PEA. Mice were weighed beforeDNBS (or vehicle) administration and immediately before killing. Tissueswere analysed 3 days after vehicle or DNBS administration. PEA (0.1-10mg·kg⁻¹) was administered once a day for three consecutive days starting24 h after the inflammatory insult (therapeutic protocol). Bars aremean±SEM of 12-15 mice for each experimental group. #P<0.001 versuscontrol (i.e. mice without intestinal inflammation). *P<0.05, **P<0.01and ***P<0.001 versus DNBS alone.

FIG. 2Q is a scatter plot measuring the % inhibition (as measured bycolon weight: length ratio) versus the amount of PEA administered fortwo populations (one via oral administration, the otherintraperitoneal).

FIG. 3A is a blank plasma chromatogram of PEA. The Y-axis measuresintensity (cps) on a scale from 0 to 365; the X-axs measured time (min)on a scale from 0 to 1.0.

FIG. 3B is a representative chromatogram of LLOQ for PEA, 2.5 ng/mL. TheY-axis measures intensity (cps) on a scale from 0 to 870; the X-axsmeasures time (min) on a scale from 0 to 1.0.

FIG. 3C is a representative chromatogram of the ULOQ for PEA, 1000ng/mL. The Y-axis measures intensity (cps) on a scale from 0 to 1.8×10⁵;the X-axs measures time (min) on a scale from 0 to 1.0.

FIG. 3D is a representative calibration curve for PEA.

FIG. 3E is a blank plasma chromatogram of PEA-prodrug I-9. The Y-axismeasures intensity (cps) on a scale from 0 to 1000; the X-axs measurestime (min) on a scale from 0 to 1.0.

FIG. 3F is a representative chromatogram of LLOQ for PEA-prodrug I-9,0.5 ng/mL. The Y-axis measures intensity (cps) on a scale from 0 to1120; the X-axs measures time (min) on a scale from 0 to 1.0.

FIG. 3G is a representative chromatogram for ULOQ for PEA prodrug I-9,1000 ng/mL. The Y-axis measures intensity (cps) on a scale from 0 to9×10⁶; the X-axs measures time (min) on a scale from 0 to 1.0.

FIG. 3H is a representative calibration curve for PEA-prodrug I-9.

FIG. 4A is a scatter plot of individual plasma concentrations of PEAafter intravenous administration of PEA in male Sprague-Dawley rats at 1mg/kg (Group 1).

FIG. 4B is a scatter plot of average plasma concentrations of PEA afterintravenous administration of PEA in male Sprague-Dawley rats at 1 mg/kg(Group 1).

FIG. 5A is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-9 at 16 mg/kg in male Sprague Dawleyrats.

FIG. 5B is a scatter plot of average plasma concentrations of PEA afteroral administration of I-9 at 16 mg/kg in male Sprague-Dawley rats.

FIG. 5C is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-6 at 19 mg/kg in male Sprague-Dawley rats(Group 2).

FIG. 5D is a scatter plot of average plasma concentrations of PEA afteroral administration of I-6.

FIG. 5E is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-5 at 19.7 mg/kg in male Sprague-Dawleyrats (Group 3).

FIG. 5F is a scatter plot of average plasma concentrations of PEA afteroral administration of I-5 at 19.7 mg/kg in male Sprague-Dawley rats(Group 3).

FIG. 5G is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-3 (PEA-Succinate-Glycerol-Di-Caprylic) at24.5 mg/kg in male Sprague-Dawley rats (Group 4).

FIG. 5H is a scatter plot of average plasma concentrations of PEA afteroral administration of I-3 (PEA-Succinate-Glycerol-Di-Caprylic) at 24.5mg/kg in male Sprague-Dawley rats (Group 4).

FIG. 5I is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-2 (PEA-BA) at 12/5 mg/kg in maleSprague-Dawley rats (Group 5).

FIG. 5J is a scatter plot of average plasma concentrations of PEA afteroral administration of I-2 (PEA-BA) at 12.5 mg/kg in male Sprague-Dawleyrats (Group 5).

FIG. 5K is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-11 at 20.7 mg/kg in Male Sprague-DawleyRats (Group 6).

FIG. 5L is a scatter plot of average plasma concentrations of PEA afteroral administration of I-11 at 20.7 mg/kg in Male Sprague-Dawley Rats(Group 6).

FIG. 5M is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-7 at 24.5 mg/kg in male Sprague-Dawleyrats (Group 7).

FIG. 5N is a scatter plot of average plasma concentrations of PEA afteroral administration of I-7 at 24.5 mg/kg in male Sprague-Dawley rats(Group 7).

FIG. 6 is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-13 (in 20% (Solutol HS15:NMP 1:1) 10%PEG400, 70% H₂O) at 24.3 mg/kg in male Sprague-Dawley rats.

FIG. 7A is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-12 (in 20% Solutol HS15:NMP (1:1), 10%PEG400, 70% H₂O) at 35.2 mg/kg in male Sprague-Dawley rats (Group 1).

FIG. 7B is a scatter plot of average plasma concentrations of PEA afteroral administration of I-12 (in 20% Solutol HS15:NMP (1:1), 10% PEG400,70% H₂O) at 35.2 mg/kg in male Sprague-Dawley rats (Group 1).

FIG. 7C is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-12 (in 0.5% Methyl Cellulose in 20%Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 35.2 mg/kg in maleSprague-Dawley rats (Group 2).

FIG. 7D is a scatter plot of average plasma concentrations of PEA afteroral administration of I-12 (in 0.5% Methyl Cellulose in 20% SolutolHS15:NMP (1:1), 10% PEG400, 70% H₂O) at 35.2 mg/kg in maleSprague-Dawley rats (Group 2).

FIG. 8A is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-15 (in 20% Solutol HS15:NMP (1:1), 10%PEG400, 70% H₂O) at 20.7 mg/kg in male Sprague-Dawley rats (Group 1).

FIG. 8B is a scatter plot of average plasma concentrations of PEA afteroral administration of I-15 (in 20% Solutol HS15:NMP (1:1), 10% PEG400,70% H₂O) at 20.7 mg/kg in male Sprague-Dawley rats (Group 1).

FIG. 8C is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-14 (in 20% Solutol HS15:NMP (1:1), 10%PEG400, 70% H₂O) at 20.7 mg/kg in male Sprague-Dawley rats (Group 1).

FIG. 8D is a scatter plot of average plasma concentrations of PEA afteroral administration of I-14 (in 20% Solutol HS15:NMP (1:1), 10% PEG400,70% H₂O) at 20.7 mg/kg in male Sprague-Dawley rats (Group 1).

FIG. 9A is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-8.

FIG. 9B is a scatter plot of average plasma concentrations of PEA afteroral administration of I-8.

FIG. 9C is a scatter plot of individual plasma concentrations of PEAafter oral administration of I-16.

FIG. 9D is a scatter plot of average plasma concentrations of PEA afteroral administration of I-16.

FIG. 10A is a scatter plot of individual plasma concentrations (ng/mL)and pharmacokinetic parameters for PEA after oral administration of I-8in 20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 4 mg/kg in maleSprague-Dawley rats.

FIG. 10B is a scatter plot of average plasma concentrations (ng/mL) andphamiacokinetic parameters for PEA after oral administration of I-8 in20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 4 mg/kg in maleSprague-Dawley rats.

FIG. 10C is a scatter plot of individual plasma concentrations (ng/mL)and pharmacokinetic parameters for PEA after oral administration of I-8in 20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 8 mg/kg in maleSprague-Dawley rats.

FIG. 10D is a scatter plot of average plasma concentrations (ng/mL) andpharmacokinetic parameters for PEA after oral administration of I-8 in20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 8 mg/kg in maleSprague-Dawley rats.

FIG. 10E is a scatter plot of individual plasma concentrations (ng/mL)and pharmacokinetic parameters for PEA after oral administration of I-8in 20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 16 mg/kg in maleSprague-Dawley rats.

FIG. 10F is a scatter plot of average plasma concentrations (ng/mL) andpharmacokinetic parameters for PEA after oral administration of I-8 in20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 16 mg/kg in maleSprague-Dawley rats.

FIG. 10G is a scatter plot of individual plasma concentrations (ng/mL)and pharmacokinetic parameters for PEA after oral administration of I-16in 20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 5.2 mg/kg in maleSprague-Dawley rats.

FIG. 10H is a scatter plot of average plasma concentrations (ng/mL) andpharmacokinetic parameters for PEA after oral administration of I-16 in20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 5.2 mg/kg in maleSprague-Dawley rats.

FIG. 10I is a scatter plot of individual plasma concentrations (ng/mL)and pharmacokinetic parameters for PEA after oral administration of I-16in 20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 10.35 mg/kg inmale Sprague-Dawley rats.

FIG. 10J is a scatter plot of average plasma concentrations (ng/mL) andpharmacokinetic parameters for PEA after oral administration of I-16 in20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 10.35 mg/kg in maleSprague-Dawley rats.

FIG. 10K is a scatter plot of individual plasma concentrations (ng/mL)and pharmacokinetic parameters for PEA after oral administration of I-16in 20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 20.7 mg/kg inmale Sprague-Dawley rats.

FIG. 10L is a scatter plot of average plasma concentrations (ng/mL) andpharmacokinetic parameters for PEA after oral administration of I-16 in20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 20.7 mg/kg in maleSprague-Dawley rats.

FIG. 11 is a bar graph illustrating the paw withdrawal latency (inseconds) as a function of amount of I-16 provided to an animal subject.The animal received either no I-16 (i.e., only the vehicle); 5 mg/kgequivalents of PEA (equivalent to 10.25 mg/kg of I-16); or 10 mg/kgequivalents of PEA (equivalent to 20.50 mg/kg of I-16).

FIG. 12A is a timeline illustrating the dosing schedule of ratsadministered I-16 to evaluate analgesic effects in rat ChronicConstriction Injury (CCI) model.

FIG. 12B is a bar graph illustrating mechanical allodvnia in rats as afunction of analgesic administered (vehicle: gabapentin; or I-16).

DEFINITIONS A. Chemical Definitions

As used herein, the following definitions shall apply unless otherwiseindicated. For purposes of this disclosure, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, Handbook of Chemistry and Physics, 75^(th) Ed. Additionally,general principles of organic chemistry are described in “OrganicChemistry”. Thomas Sorrell, University Science Books, Sausalito: 1999,and “March's Advanced Organic Chemistry”, 5^(th) Ed., Ed.: Smith, M. B.and March, J., John Wiley & Sons, New York: 2001, the entire contents ofwhich are hereby incorporated by reference.

Aliphatic: As used herein, “aliphatic” means a straight-chain (i.e.,unbranched) or branched, substituted or unsubstituted hydrocarbon chainthat is completely saturated or that contains one or more units ofunsaturation, or a monocyclic hydrocarbon, bicyclic hydrocarbon, orpolycyclic hydrocarbon that is completely saturated or that contains oneor more units of unsaturation that has a single point of attachment tothe rest of the molecule. Unless otherwise specified, aliphatic groupscontain 1-100 aliphatic carbon atoms. In some embodiments, aliphaticgroups contain 1-20 aliphatic carbon atoms. In other embodiments,aliphatic groups contain 1-10 aliphatic carbon atoms. In still otherembodiments, aliphatic groups contain 1-5 aliphatic carbon atoms, and inyet other embodiments, aliphatic groups contain 1, 2, 3, or 4 aliphaticcarbon atoms. Suitable aliphatic groups include, hut are not limited to,linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynylgroups and hybrids thereof.

Alkyl: As used herein, the term “alkyl” is given its ordinary meaning inthe art and may include saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. In some embodiments, alkyl has 1-100 carbonatoms. In certain embodiments, a straight chain or branched chain alkylhas about 1-20 carbon atoms in its backbone (e.g., C₁-C₂₀ for straightchain, C₂-C₂₀ for branched chain), and alternatively, about 1-10. Insome embodiments, a cycloalkyl ring has from about 3-10 carbon atoms intheir ring structure where such rings are monocyclic or bicyclic, andalternatively about 5, 6 or 7 carbons in the ring structure. In someembodiments, an alkyl group may be a lower alkyl group, wherein a loweralkyl group comprises 1-4 carbon atoms (e.g., C₁-C₄ for straight chainlower alkyls).

Alkenyl: As used herein, the term “alkenyl” refers to an alkyl group, asdefined herein, having one or more double bonds.

Alkynyl: As used herein, the term “alkynyl” refers to an alkyl group, asdefined herein, having one or more triple bonds.

Protecting Group: The phrase “protecting group,” as used herein, refersto temporary substituents which protect a potentially reactivefunctional group from undesired chemical transformations. Examples ofsuch protecting groups include esters of carboxylic acids, silyl ethersof alcohols, and acetals and ketals of aldehydes and ketones,respectively. A “Si protecting group” is a protecting group comprising aSi atom, such as Si-trialkyl (e.g., trimethylsilyl, tributylsilyl,t-butyldimethylsilyl), Si-triaryl, Si-alkyl-diphenyl (e.g.,t-butyldiphenylsilyl), or Si-aryl-dialkyl (e.g., Si-phenyldialkyl).Generally, a Si protecting group is attached to an oxygen atom. Thefield of protecting group chemistry has been reviewed (Greene, T. W.;Wuts, P. G. M.

Protective Groups in Organic Synthesis, 2nd ed.; Wiley: New York, 1991).Such protecting groups (and associated protected moieties) are describedin detail below. Protected hydroxyl groups are well known in the art andinclude those described in detail in Protecting Groups in OrganicSynthesis, T. W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley &Sons, 1999, the entirety of which is incorporated herein by reference.Examples of suitably protected hydroxyl groups further include, but arenot limited to, esters, carbonates, sulfonates, allyl ethers, ethers,silyl ethers, alkyl ethers, arylalkyl ethers, and alkoxyalkyl ethers.Examples of suitable esters include formates, acetates, proprionates,pentanoates, crotonates, and benzoates. Specific examples of suitableesters include formate, benzoyl formate, chloroacetate,trifluoroacetate, methoxyacetate, triphenylmethoxy acetate,p-chlorophenoxyacetate, 3-phenylpropionate, 4-oxopentanoate,4,4-(ethylenedithio)pentanoate, pivaloate (trimethylacetate), crotonate,4-methoxy-crotonate, benzoate, p-benzylbenzoate,2,4,6-trimethylbenzoate. Examples of suitable carbonates include9-fluorenylmethyl, ethyl, 2,2,2-trichloroethyl, 2-(trimethylsilyl)ethyl,2-(phenylsulfonyl)ethyl, vinyl, allyl, and p-nitrobenzyl carbonate.Examples of suitable silyl ethers include trimethylsilyl, triethylsilyl,t-butyldimethylsilyl, t-butyldiphenylsilyl, triisopropylsilyl ether, andother trialkylsilyl ethers. Examples of suitable alkyl ethers includemethyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, trityl, t-butyl,and allyl ether, or derivatives thereof. Alkoxyalkyl ethers includeacetals such as methoxymethyl, methylthiomethyl,(2-methoxyethoxy)methyl, benzyloxymethyl,beta-(trimethylsilyl)ethoxymethyl, and tetrahydropyran-2-yl ether.Examples of suitable arylalkyl ethers include benzyl, p-methoxybenzyl(MPM), 3,4-dimethoxy benzyl, O-nitrobenzyl, p-nitrobenzyl, p-halobenzyl,2,6-dichlorobenzyl, p-cyanobenzyl, 2- and 4-picolyl ethers.

Protected amines are well known in the art and include those describedin detail in Greene (1999). Suitable mono-protected amines furtherinclude, but are not limited to, aralkylamines, carbamates, allylamines, amides, and the like. Examples of suitable mono-protected aminomoieties include t-butyloxycarbonylamino (—NHBOC),ethyloxycarbonylamino, methyloxycarbonylamino,trichloroethyloxycarbonylamino, allyloxycarbonylamino (—NHAlloc),benzyloxocarbonylamino (—NHCBZ), allylamino, benzylamino (—NHBn),fluorenylmethylcarbonyl (—NHFmoc), formamido, acetamido,chloroacetamido, dichloroacetamido, trichloroacetamido, phenylacetamido,trifluoroacetamido, benzamido, t-butyldiphenylsilyl, and the like.Suitable di-protected amines include amines that are substituted withtwo substituents independently selected from those described above asmono-protected amines, and further include cyclic imides, such asphthalimide, maleimide, succinimide, and the like. Suitable di-protectedamines also include pyrroles and the like,2,2,5,5-tetramethyl-[1,2,5]azadisilolidine and the like, and azide.

Protected aldehydes are well known in the art and include thosedescribed in detail in Greene (1999). Suitable protected aldehydesfurther include, but are not limited to, acyclic acetals, cyclicacetals, hydrazones, imines, and the like. Examples of such groupsinclude dimethyl acetal, diethyl acetal, diisopropyl acetal, dibenzylacetal, bis(2-nitrobenzyl) acetal, 1,3-dioxanes, 1,3-dioxolanes,semicarbazones, and derivatives thereof.

Protected carboxylic acids are well known in the art and include thosedescribed in detail in Greene (1999). Suitable protected carboxylicacids further include, but are not limited to, optionally substitutedC₁₋₆ aliphatic esters, optionally substituted aryl esters, silyl esters,activated esters, amides, hydrazides, and the like. Examples of suchester groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl,benzyl, and phenyl ester, wherein each group is optionally substituted.Additional suitable protected carboxylic acids include oxazolines andortho esters.

Protected thiols are well known in the art and include those describedin detail in Greene (1999). Suitable protected thiols further include,but are not limited to, disulfides, thioethers, silyl thioethers,thioesters, thiocarbonates, and thiocarbamates, and the like. Examplesof such groups include, but are not limited to, alkyl thioethers, benzyland substituted benzyl thioethers, triphenylmethyl thioethers, andtnchloroethoxycarbonyl thioester, to name but a few.

Substitution: As described herein, compounds of the disclosure maycontain optionally substituted and/or substituted moieties. In general,the term “substituted,” whether preceded by the term “optionally” ornot, means that one or more hydrogens of the designated moiety arereplaced with a suitable substituent. Unless otherwise indicated, an“optionally substituted” group may have a suitable substituent at eachsubstitutable position of the group, and when more than one position inany given structure may be substituted with more than one substituentselected from a specified group, the substituent may be either the sameor different at every position. Combinations of substituents envisionedby this disclosure are preferably those that result in the formation ofstable or chemically feasible compounds. The term “stable,” as usedherein, refers to compounds that are not substantially altered whensubjected to conditions to allow for their production, detection, and,in certain embodiments, their recovery, purification, and use for one ormore of the purposes disclosed herein.

Suitable monovalent substituents include halogen; —(CH₂)₀₋₄R^(∘);—(CH₂)₀₋₄OR^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄Ph, which may be substituted with R^(∘);—(CH₂)₀₋₄(CH₂)₀₋₁Ph which may be substituted with R^(∘); —CH═CHPh, whichmay be substituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may besubstituted with R^(∘); —NO₂; —CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂;—(CH₂)₀₋₄N(R^(∘))C(O)R^(∘); —N(R^(∘))C(S)R^(∘);—(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘) ₂;—(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR^(∘)—, —SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘);—(CH₂)₀₋₄C(O)NR^(∘) ₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘),—(CH₂)₀₋₄O C(O)NR^(∘) ₂; —C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘);—C(O)CH₂C(O)R^(∘); —C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘):—(CH₂)₀₋₄S(O)₂R^(∘); —(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘);—S(O)₂NR^(∘) ₂; —(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂;—N(R^(∘))S(O)₂R^(∘); —N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘);—P(O)R^(∘) ₂; —OP(O)R^(∘) ₂; —OP(O)(OR^(∘))₂; —SiR^(∘) ₃; —OSiR^(∘) ₃;—(C₁₋₄ straight or branched)alkylene)O—N(R^(∘))₂; or —(C₁₋₄ straight orbranched)alkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substitutedas defined below and is independently hydrogen, C₁₋₆, aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6 membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(•), -(haloR^(•)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(•), —(CH₂)₀₋₂CH(OR^(•))₂; —O(haloR^(•)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(•), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(•),—(CH₂)₀₋₂SR^(•), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(•),—(CH₂)₀₋₂NR^(•)2, —NO₂, —SiR^(•) ₃, —C(O)SR^(•), —(C₁₋₄ straight orbranched alkylene)C(O)OR^(•), or —SSR^(•) wherein each R^(•) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently selected from C₁₋₄ aliphatic.—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR. —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,R^(•). -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —CN, —C(O)OH,—C(O)OR^(•), —NHR^(•), —NR^(•) ₂, or —NO₂, wherein each R^(•) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

In some embodiments, suitable substituents on a substitutable nitrogeninclude —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†), —C(O)C(O)R^(†),—C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂, —C(S)NR^(†) ₂,—C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein each R^(†) isindependently hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†) taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. [105]

Suitable substituents on the aliphatic group of R^(†) are independentlyhalogen. —R^(•), -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —CN,—C(O)OH, —C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein eachR^(•) is unsubstituted or where preceded by “halo” is substituted onlywith one or more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6- membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention.

B. Other Definitions

Administration: As used herein, the term “administration” typicallyrefers to the administration of a composition to a subject or system.Those of ordinary skill in the art will be aware of a variety of routesthat may, in appropriate circumstances, be utilized for administrationto a subject, for example a human. For example, in some embodiments,administration may be ocular, oral, parenteral, topical, etc.. In someparticular embodiments, administration may be bronchial (e.g., bybronchial instillation), buccal, dermal (which may be or comprise, forexample, one or more of topical to the dermis, mtradermal, interdermal,transdermal, etc), enteral, intra-arterial, intradermal, mtragastric,intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal,intravenous, intraventricular, within a specific organ (e. g.intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual,topical, tracheal (e.g., by intratracheal instillation), vaginal,vitreal, etc. In some embodiments, administration may involve dosingthat is intermittent (e.g., a plurality of doses separated in time)and/or periodic (e.g., individual doses separated by a common period oftime) dosing. In some embodiments, administration may involve continuousdosing (e.g., perfusion) for at least a selected penod of time.

Agent: In general, the term “agent”, as used herein, may be used torefer to a compound or entity of any chemical class including, forexample, a polypeptide, nucleic acid, saccharide, lipid, small molecule,metal, or combination or complex thereof. In appropriate circumstances,as will be clear from context to those skilled in the art, the tertn maybe utilized to refer to an entity that is or comprises a cell ororganism, or a fraction, extract, or component thereof. Alternatively oradditionally, as context will make clear, the term may be used to referto a natural product in that it is found in and/or is obtained fromnature. In some instances, again as will be clear from context, the termmay be used to refer to one or more entities that is man-made in that itis designed, engineered, and/or produced through action of the hand ofman and/or is not found in nature. In some embodiments, an agent may beutilized in isolated or pure form; in some embodiments, an agent may beutilized in crude form. In some embodiments, potential agents may beprovided as collections or libraries, for example that may be screenedto identify or characterize active agents within them. In some cases,the term “agent” may refer to a compound or entity that is or comprisesa polymer; in some cases, the term may refer to a compound or entitythat comprises one or more polymeric moieties. In some embodiments, theterm “agent” may refer to a compound or entity that is not a polymerand/or is substantially free of any polymer and/or of one or moreparticular polymeric moieties. In some embodiments, the term may referto a compound or entity that lacks or is substantially free of anypolymeric moiety.

Agonist: Those skilled in the art will appreciate that the term“agonist” may be used to refer to an agent condition, or event whosepresence, level, degree, type, or form correlates with increased levelor activity of another agent (i.e., the agonized agent). In general, anagonist may be or include an agent of any chemical class including, forexample, small molecules, polypeptides, nucleic acids, carbohydrates,lipids, metals, and/or any other entity that shows the relevantactivating activity. In some embodiments, an agonist may be direct (inwhich case it exerts its influence directly upon its target); in someembodiments, an agonist may be indirect (in which case it exerts itsinfluence by other than binding to its target; e.g., by interacting witha regulator of the target, so that level or activity of the target isaltered).

Animal: As used herein refers to any member of the animal kingdom. Insome embodiments, “animal” refers to humans, of either sex and at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal, geneticallyengineered animal, and/or a clone.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Associated with: Two events or entities are “associated” with oneanother, as that term is used herein, if the presence, level and/or formof one is correlated with that of the other. For example, a particularentity (e.g., polypeptide, genetic signature, metabolite, microbe, etc)is considered to be associated with a particular disease, disorder, orcondition, if its presence, level and/or form correlates with incidenceof and/or susceptibility to the disease, disorder, or condition (e.g.,across a relevant population). In some embodiments, two or more entitiesare physically “associated” with one another if they interact, directlyor indirectly, so that they are and/or remain in physical proximity withone another. In some embodiments, two or more entities that arephysically associated with one another are covalently linked to oneanother; in some embodiments, two or more entities that are physicallyassociated with one another are not covalently linked to one another butare non-covalently associated, for example by means of hydrogen bonds,van der Waals interaction, hydrophobic interactions, magnetism, andcombinations thereof.

Carrier: as used herein, refers to a diluent, adjuvant, excipient, orvehicle with which a composition is administered. In some exemplaryembodiments, carriers can include sterile liquids, such as, for example,water and oils, including oils of petroleum, animal, vegetable orsynthetic origin, such as, for example, peanut oil, soybean oil, mineraloil, sesame oil and the like. In some embodiments, carriers are orinclude one or more solid components.

Comparable: As used herein, the term “comparable” refers to two or moreagents, entities, situations, sets of conditions, etc., that may not beidentical to one another but that are sufficiently similar to permitcomparison there between so that one skilled in the art will appreciatethat conclusions may reasonably be drawn based on differences orsimilarities observed. In some embodiments, comparable sets ofconditions, circumstances, individuals, or populations are characterizedby a plurality of substantially identical features and one or a smallnumber of varied features. Those of ordinary skill in the art willunderstand, in context, what degree of identity is required in any givencircumstance for two or more such agents, entities, situations, sets ofconditions, etc to be considered comparable. For example, those ofordinary skill in the art will appreciate that sets of circumstances,individuals, or populations are comparable to one another whencharacterized by a sufficient number and type of substantially identicalfeatures to warrant a reasonable conclusion that differences in resultsobtained or phenomena observed under or with different sets ofcircumstances, individuals, or populations are caused by or indicativeof the variation in those features that are varied.

Composition: Those skilled in the art will appreciate that the term“composition” may be used to refer to a discrete physical entity thatcomprises one or more specified components. In general, unless otherwisespecified, a composition may be of any form—e.g., gas, gel, liquid,solid, etc.

Comprising: A composition or method described herein as “comprising” oneor more named elements or steps is open-ended, meaning that the namedelements or steps are essential, but other elements or steps may beadded within the scope of the composition or method. To avoid prolixity,it is also understood that any composition or method described as“comprising” (or which “comprises”) one or more named elements or stepsalso describes the corresponding, more limited composition or method“consisting essentially of (or which “consists essentially of”) the samenamed elements or steps, meaning that the composition or method includesthe named essential elements or steps and may also include additionalelements or steps that do not materially affect the basic and novelcharacteristic(s) of the composition or method. It is also understoodthat any composition or method described herein as “comprising” or“consisting essentially of” one or more named elements or steps alsodescribes the corresponding, more limited, and closed-ended compositionor method “consisting of (or “consists of”) the named elements or stepsto the exclusion of any other unnamed element or step. In anycomposition or method disclosed herein, known or disclosed equivalentsof any named essential element or step may be substituted for thatelement or step.

Dosage form or unit dosage form: Those skilled in the art willappreciate that the term “dosage form” may be used to refer to aphysically discrete unit of an active agent (e.g., a therapeutic ordiagnostic agent) for administration to a subject. Typically, each suchunit contains a predetermined quantity′ of active agent. In someembodiments, such quantity is a unit dosage amount (or a whole fractionthereof) appropriate for administration in accordance with a dosingregimen that has been determined to correlate with a desired orbeneficial outcome when administered to a relevant population (i.e.,with a therapeutic dosing regimen). Those of ordinary skill in the artappreciate that the total amount of a therapeutic composition or agentadministered to a particular subject is determined by one or moreattending physicians and may involve administration of multiple dosageforms.

Dosing regimen: Those skilled in the art will appreciate that the term“dosing regimen” may be used to refer to a set of unit doses (typicallymore than one) that are administered individually to a subject,typically separated by periods of time. In some embodiments, a giventherapeutic agent has a recommended dosing regimen, which may involveone or more doses. In some embodiments, a dosing regimen comprises aplurality of doses each of which is separated in time from other doses.In some embodiments, individual doses are separated from one another bya time period of the same length; in some embodiments, a dosing regimencomprises a plurality of doses and at least two different time periodsseparating individual doses. In some embodiments, all doses within adosing regimen are of the same unit dose amount. In some embodiments,different doses within a dosing regimen are of different amounts. Insome embodiments, a dosing regimen comprises a first dose in a firstdose amount, followed by one or more additional doses in a second doseamount different from the first dose amount. In some embodiments, adosing regimen comprises a first dose in a first dose amount, followedby one or more additional doses in a second dose amount same as thefirst dose amount. In some embodiments, a dosing regimen is correlatedwith a desired or beneficial outcome when administered across a relevantpopulation (i.e., is a therapeutic dosing regimen).

Encapsulated: The term “encapsulated” is used herein to refer tosubstances that are completely surrounded by another material.

Excipient: as used herein, refers to a non-therapeutic agent that may beincluded in a pharmaceutical composition, for example to provide orcontribute to a desired consistency or stabilizing effect. Suitablepharmaceutical excipients include, for example, starch, glucose,lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodiumstearate, glycerol monostearate, talc, sodium chloride, dried skim milk,glycerol, propylene, glycol, water, ethanol and the like.

Gel: As used herein, the term “gel” refers to viscoelastic materialswhose rheological properties distinguish them from solutions, solids,etc. In some embodiments, a composition is considered to be a gel if itsstorage modulus (G′) is larger than its modulus (G″). In someembodiments, a composition is considered to be a gel if there arechemical or physical cross-linked networks in solution, which isdistinguished from entangled molecules in viscous solution.

“Improved,” “increased” or “reduced”: As used herein, these terms, orgrammatically comparable comparative terms, indicate values that arerelative to a comparable reference measurement. For example, in someembodiments, an assessed value achieved with an agent of interest may be“improved” relative to that obtained with a comparable reference agent.Alternatively or additionally, in some embodiments, an assessed valueachieved in a subject or system of interest may be “improved” relativeto that obtained in the same subject or system under differentconditions (e.g., prior to or after an event such as administration ofan agent of interest), or in a different, comparable subject (e.g., in acomparable subject or system that differs from the subject or system ofinterest in presence of one or more indicators of a particular disease,disorder or condition of interest, or in prior exposure to a conditionor agent, etc). In some embodiments, comparative terms refer tostatistically relevant differences (e.g., that are of a prevalenceand/or magnitude sufficient to achieve statistical relevance). Thoseskilled in the art will be aware, or will readily be able to determine,in a given context, a degree and/or prevalence of difference that isrequired or sufficient to achieve such statistical significance.

Intraperitoneal: The phrases “intraperitoneal administration” and“administered intraperitoneally” as used herein have theirart-understood meaning referring to administration of a compound orcomposition into the peritoneum of a subject.

Moiety: Those skilled in the art will appreciate that a “moiety” is adefined chemical group or entity with a particular structure and/or oractivity, as described herein.

Oral: The phrases “oral administration” and “administered orally” asused herein have their art-understood meaning referring toadministration by mouth of a compound or composition.

Parent N-acylethanolamide compound: A “parent” N-acylethanolamidecompound, for purposes of the present disclosure, is a compound relativeto which the present disclosure provides derivatives (e.g., to provide acompound of described herein). Typically, a parent N-acylethanolamidecompound has a structure as set forth below:

wherein R¹ is C₁₋₄₀ aliphatic.

In some embodiments, R^(x) is C₁₋₄₀ aliphatic. In some embodiments, leis C₁₋₃₅ aliphatic. In some embodiments, R^(x) is C₁₋₃₀ aliphatic. Insome embodiments, R^(x) is C₁₋₂₅ aliphatic. In some embodiments, R^(x)is C₁₋₂₀ aliphatic. In some embodiments, R^(x) is C₁₋₁₅ aliphatic. Insome embodiments, R^(x) is C₁₋₁₀ aliphatic. In some embodiments R^(x) isC₁₋₅ aliphatic. In some embodiments, R^(x) is C₅₋₃₀ aliphatic. In someembodiments, R^(x) is C₁₀₋₁₅ aliphatic. In some embodiments, R^(x) isC₁₀₋₂₀ aliphatic. In some embodiments, le is C₅₋₁₅ aliphatic. In someembodiments, R^(x) is C₁₅₋₂₅ aliphatic. In some embodiments, a parentN-acylethanolamide compound is derived from a fatty acid selected fromthe group consisting of myristoleic acid, palmitoleic acid, sapienicacid, oleic acid, elaidic acid, vaccenic acid, linoleic acid,linoelaidic acid, a-linolenic acid, arachidonic acid, eicosapentaenoicacid, erucic acid, docosahexaenoic acid, caprylic acid, capric acid,lauric acid, myrishc acid, palmitic acid, stearic acid, arachidic acid,behemc acid, lignocenc acid, and cerotic acid. In some embodiments, aparent N-acylethanolamide compound is selected from the group consistingof N-palmitoylethanolamide, N-oleoylethanolamide, andN-arachidonoylethanolamide. In some embodiments, a parentN-acylethanolamide compound is N-palmitoylethanolamide. In someembodiments, a parent N-acylethanolamide compound isN-oleoylethanolamide. In some embodiments, a parent N-acylethanolamidecompound is N-arachidonoylethanolamide.

Parenteral: The phrases “parenteral administration” and “administeredparenterally” as used herein have their art-understood meaning referringto modes of administration other than enteral and topicaladministration, usually by injection, and include, without limitation,intravenous, intramuscular, intra.arterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid,mtraspinal, and intrastemal injection and infusion.

Patient: As used herein, the term “patient” refers to any organism towhich a provided composition is or may be administered, e.g., forexperimental, diagnostic, prophylactic, cosmetic, and/or therapeuticpurposes. Typical patients include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and/or humans). In some embodiments,a patient is a human. In some embodiments, a patient is suffering fromor susceptible to one or more disorders or conditions. In someembodiments, a patient displays one or more symptoms of a disorder orcondition. In some embodiments, a patient has been diagnosed with one ormore disorders or conditions. In some embodiments, the disorder orcondition is or includes cancer, or presence of one or more tumors. Insome embodiments, the patient is receiving or has received certaintherapy to diagnose and/or to treat a disease, disorder, or condition.

Pharmaceutical composition: As used herein, the term “pharmaceuticalcomposition” refers to an active agent, formulated together with one ormore pharmaceutically acceptable carriers. In some embodiments, activeagent is present in unit dose amount appropriate for administration in atherapeutic regimen that shows a statistically significant probabilityof achieving a predetermined therapeutic effect when administered to arelevant population. In some embodiments, pharmaceutical compositionsmay be specially formulated for administration in solid or liquid form,including those adapted for the following: oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),tablets, e.g., those targeted for buccal, sublingual, and systemicabsorption, boluses, powders, granules, pastes for application to thetongue; parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation;topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin, lungs, or oralcavity; intravaginally or intrarectally, for example, as a pessary,cream, or foam; sublingually; ocularly; transdermally; or nasally,pulmonary, and to other mucosal surfaces.

Pharmaceutically acceptable: As used herein, the phrase“pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Pharmaceutically acceptable carrier: As used herein, the term“pharmaceutically acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

Pharmaceutically acceptable salt: The term “pharmaceutically acceptablesalt”, as used herein, refers to salts of such compounds that areappropriate for use in pharmaceutical contexts, i.e., salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, S. M. Berge, et al. describespharmaceutically acceptable salts in detail in J. PharmaceuticalSciences, 66: 1-19 (1977). In some embodiments, pharmaceuticallyacceptable salts include, but are not limited to, nontoxic acid additionsalts, which are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,maleic acid, tartaric acid, citric acid, succinic acid or malomc acid orby using other methods used in the art such as ion exchange. In someembodiments, pharmaceutically acceptable salts include, but are notlimited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate,benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate,citrate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like. In someembodiments, pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms,sulfonate and aryl sulfonate.

Predetermined: By predetermined is meant deliberately selected, forexample as opposed to randomly occurring or achieved.

Prevent or prevention: as used herein when used in connection with theoccurrence of a disease, disorder, and/or condition, refers to reducingthe risk of developing the disease, disorder and/or condition and/or todelaying onset of one or more characteristics or symptoms of thedisease, disorder or condition. Prevention may be considered completewhen onset of a disease, disorder or condition has been delayed for apredefined period of time.

As used herein, the term “prodrug” refers to a compound that is a drugprecursor which, following administration, released the drug in vivo viaa chemical or physiological process (e.g., a prodrug released the drugupon reaching physiological pH or through enzyme action is converted tothe desired drug form).

Reference: As used herein describes a standard or control relative towhich a comparison is performed. For example, in some embodiments, anagent, animal, individual, population, sample, sequence or value ofinterest is compared with a reference or control agent, animal,individual, population, sample, sequence or value. In some embodiments,a reference or control is tested and/or determined substantiallysimultaneously with the testing or determination of interest. In someembodiments, a reference or control is a historical reference orcontrol, optionally embodied in a tangible medium. Typically, as wouldbe understood by those skilled in the art, a reference or control isdetermined or characterized under comparable conditions or circumstancesto those under assessment. Those skilled in the art will appreciate whensufficient similarities are present to justify reliance on and/orcomparison to a particular possible reference or control.

Subject: As used herein, the term “subject” or “test subject” refers toany organism to which a provided compound or composition is administeredin accordance with the present disclosure e.g., for experimental,diagnostic, prophylactic, and/or therapeutic purposes. Typical subjectsinclude animals (e.g., mammals such as mice, rats, rabbits, non-humanprimates, and humans; insects; worms; etc.) and plants. In someembodiments, a subject may be suffering from, and/or susceptible to adisease, disorder, and/or condition.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with and/or displays oneor more symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition is one who has a higher risk of developingthe disease, disorder, and/or condition than does a member of thegeneral public. In some embodiments, an individual who is susceptible toa disease, disorder and/or condition may not have been diagnosed withthe disease, disorder, and/or condition. In some embodiments, anindividual who is susceptible to a disease, disorder, and/or conditionmay exhibit symptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition may not exhibit symptoms of the disease, disorder,and/or condition. In some embodiments, an individual who is susceptibleto a disease, disorder, and/or condition will develop the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will not developthe disease, disorder, and/or condition.

Systemic: The phrases “systemic administration,” “administeredsystemically,” “peripheral administration,” and “administeredperipherally” as used herein have their art-understood meaning referringto administration of a compound or composition such that it enters therecipient's system.

Tautomeric forms: The phrase “tautomeric forms,” as used herein, is usedto describe different isomeric forms of organic compounds that arecapable of facile interconversion. Tautomers may be characterized by theformal migration of a hydrogen atom or proton, accompanied by a switchof a single bond and adjacent double bond. In some embodiments,tautomers may result from prototropic tautomerism (i.e., the relocationof a proton). In some embodiments, tautomers may result from valencetautomerism (i.e., the rapid reorganization of bonding electrons). Allsuch tautomeric forms are intended to be included within the scope ofthe present disclosure. In some embodiments, tautomeric forms of acompound exist in mobile equilibrium with each other, so that attemptsto prepare the separate substances results in the formation of amixture. In some embodiments, tautomeric forms of a compound areseparable and isolatable compounds. In some embodiments of thedisclosure, chemical compositions may be provided that are or includepure preparations of a single tautomeric form of a compound. In someembodiments, chemical compositions may be provided as mixtures of two ormore tautomeric forms of a compound. In certain embodiments, suchmixtures contain equal amounts of different tautomeric forms; in certainembodiments, such mixtures contain different amounts of at least twodifferent tautomeric forms of a compound. In some embodiments of thedisclosure, chemical compositions may contain all tautomeric forms of acompound. In some embodiments of the disclosure, chemical compositionsmay contain less than all tautomeric forms of a compound. In someembodiments of the disclosure, chemical compositions may contain one ormore tautomeric forms of a compound in amounts that vary over time as aresult of interconversion. In some embodiments of the disclosure, thetautomerism is keto-enol tautomerism. One of skill in the chemical artswould recognize that a keto-enol tautomer can be “trapped” (i.e.,chemically modified such that it remains in the “enol” form) using anysuitable reagent known in the chemical arts in to provide an endderivative that may subsequently be isolated using one or more suitabletechniques known in the art. Unless otherwise indicated, the presentdisclosure encompasses all tautomeric forms of relevant compounds,whether in pure form or in admixture with one another.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto an agent that, when administered to a subject, has a therapeuticeffect and/or elicits a desired biological and/or pharmacologicaleffect. In some embodiments, a therapeutic agent is any substance thatcan be used to alleviate, ameliorate, relieve, inhibit, prevent, delayonset of, reduce severity of, and/or reduce incidence of one or moresymptoms or features of a disease, disorder, and/or condition.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of a substance (e.g.,a therapeutic agent, composition, and/or formulation) that elicits adesired biological response when administered as part of a therapeuticregimen. In some embodiments, a therapeutically effective amount of asubstance is an amount that is sufficient, when administered to asubject suffering from or susceptible to a disease, disorder, and/orcondition, to treat, diagnose, prevent, and/or delay the onset of thedisease, disorder, and/or condition. As will be appreciated by those ofordinary skill in this art, the effective amount of a substance may varydepending on such factors as the desired biological endpoint, thesubstance to be delivered, the target cell or tissue, etc. For example,the effective amount of compound in a formulation to treat a disease,disorder, and/or condition is the amount that alleviates, ameliorates,relieves, inhibits, prevents, delays onset of, reduces severity ofand/or reduces incidence of one or more symptoms or features of thedisease, disorder, and/or condition. In some embodiments, atherapeutically effective amount is administered in a single dose; insome embodiments, multiple unit doses are required to deliver atherapeutically effective amount.

Therapeutic regimen: A “therapeutic regimen”, as that term is usedherein, refers to a dosing regimen whose administration across arelevant population may be correlated with a desired or beneficialtherapeutic outcome.

Treat: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof, and/or reduce incidence of one or more symptoms or features of adisease, disorder, and/or condition. Treatment may be administered to asubject who does not exhibit signs of a disease, disorder, and/orcondition. In some embodiments, treatment may be administered to asubject who exhibits only early signs of the disease, disorder, and/orcondition, for example for the purpose of decreasing the risk ofdeveloping pathology associated with the disease, disorder, and/orcondition.

Unit dose: The expression “unit dose” as used herein refers to an amountadministered as a single dose and/or in a physically discrete unit of apharmaceutical composition. In many embodiments, a unit dose contains apredetermined quantity of an active agent. In some embodiments, a unitdose contains an entire single dose of the agent. In some embodiments,more than one unit dose is administered to achieve a total single dose.In some embodiments, administration of multiple unit doses is required,or expected to be required, in order to achieve an intended effect. Aunit dose may be, for example, a volume of liquid (e.g., an acceptablecarrier) containing a predetermined quantity of one or more therapeuticagents, a predetermined amount of one or more therapeutic agents insolid form, a sustained release formulation or drug delivery devicecontaining a predetermined amount of one or more therapeutic agents,etc. It will be appreciated that a unit dose may be present in aformulation that includes any of a variety of components in addition tothe therapeutic agent(s). For example, acceptable carriers (e.g.,pharmaceutically acceptable carriers), diluents, stabilizers, buffers,preservatives, etc., may be included as described infra. It will beappreciated by those skilled in the art, in many embodiments, a totalappropriate daily dosage of a particular therapeutic agent may comprisea portion, or a plurality, of unit doses, and may be decided, forexample, by the attending physician within the scope of sound medicaljudgment. In some embodiments, the specific effective dose level for anyparticular subject or organism may depend upon a variety of factorsincluding the disorder being treated and the severity of the disorder;activity of specific active compound employed; specific compositionemployed; age, body weight, general health, sex and diet of the subject;time of administration, and rate of excretion of the specific activecompound employed; duration of the treatment; drugs and/or additionaltherapies used in combination or coincidental with specific compound(s)employed, and like factors well known in the medical arts.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

N-acylethanolamides have shown promise in treatment of various diseases,disorders, and conditions. In some embodiments, one or more compoundsprovided herein may be useful in treatment of such diseases, disordersand conditions. In some embodiments, one or more provided compounds maybe useful, for example, in treatment of one or more neurologic diseases,disorders or conditions. In some embodiments, one or more compoundsprovided herein may be useful, for example, in treatment of pain,anxiety, depression, schizophrenia, amyotrophic lateral sclerosis,multiple sclerosis, Parkinson's disease, Alzheimer's disease,Huntington's disease, neuropathic pain, cerebral ischemia, epilepsy,appetite loss, dental pain, osteoarthritis, reduced gastrointestinalmotility, cancer, glaucoma, atopic dermatitis, respiratory infection,post-traumatic stress disorder, obesity, insomnia, sleepiness, and/orIrritable Bowel Syndrome with Diarrhea (IBS-D).

In some embodiments, one or more compounds provided herein may be usefulin reducing gastrointestinal motility in a subject. In some embodiments,one or more compounds provided herein may be useful in reducing cancercell proliferation in a subject or in a biological sample. In someembodiments, one or more compounds provided herein may be useful ininducing lipolysis in a patient or in a biological sample.

The success of N-acylethanolamides, as well as their sub-optimalpharmacological properties, has led to the development of derivatives,compositions, and prodrugs. Certain N-acylethanolamide derivativesdisplay improved pharmacological properties. For example, polyethyleneglycol derivatives of N-acylethanolamides result in improvedphysico-chemical properties for the treatment of inflammatory and itch-or pain-associated disorders. See, for example, US 2015/0157733 A1.

However, such derivatives, compositions, and prodrugs have failed toproduce N-acylethanolamides with improved oral bioavailability suitablefor oral administration at high dosages. As a result, currentadministration of N-acylethanolamides must be parenteral, oftenintravenous. See, for example, Vacondio, F. et al. “Amino AcidDerivatives as Palmitoylethanolamide Prodrugs: Synthesis, In VitroMetabolism, and In Vivo Plasma Profile in Rats” PLoS' One 2015, 10(6),e0128699.

In some embodiments, the present invention provides derivatives ofN-acylethanolamides with desirable pharmacological properties, forexample, which may be or include one or more improved propertiesrelative to appropriate N-acylethanolamide reference compounds (e.g.,the parent compound of a particular derivative). In certain embodiments,the present disclosure provides derivatives of parent N-acylethanolamidecompounds that are characterized by one or more suboptimalpharmacological properties.

In certain embodiments, provided N-acylethanolamide derivative compoundsmay be characterized by one or more of the properties of increased oralbioavailability, increased cell permeability, increased watersolubility, reduced first-pass effect, increased stability, activetransport by intestinal transporters, or avoidance of effluxtransporters, when compared to N-acylethanolamide reference compounds(e.g., the parent N-acylethanolamide compound of a particularderivative). In some embodiments, provided N-acylethanolamide derivativecompounds may be characterized by increased oral bioavailability whencompared to N-acylethanolamide reference compounds (e.g., the parentN-acylethanolamide compound of a particular derivative). In someembodiments, the present invention provides N-acylethanolamidederivative compounds that are administered orally. In some embodiments,the present invention provides N-aclethanolamide derivative compoundsthat may be administered orally at high dosages.

Furthermore, administration of provided N-acylethanolamide derivativecompounds may lead to its ability to function in the treatment ofdiseases, disorders, or conditions.

Provided N-Acylethanolamide Derivatives

In some embodiments, a compound for use in accordance with the presentdisclosure is one wherein an N-acylethanolamide is conjugated to amoiety selected from the group consisting of phosphate, butyric acid,glycerol, succinate, capryhc acid, gluconoic acid, eicosapentaeonoicacid, linoleic acid, succinate, and sucrose moieties, and combinationsthereof. In some embodiments, an N-acylethanolamide is conjugated to oneor more such moieties through use of a linker moiety. In someembodiments, an N-acylethanolamide is conjugated to two or more suchmoieties. In some embodiments, an N-acylethanolamide is conjugated toone, two, or three such moieties.

In some embodiments, a provided compound has a chemical structurerepresented by formula I-a:

X₁-X₂

-   -   or a pharmaceutically acceptable salt thereof; wherein    -   X₁ is an N-acylethanolamide; and    -   X₂ is a moiety conjugated to the N-acylethanolamide.

In some embodiments, X₁ is selected from the group consisting ofN-palmitoylethanolamide, N-oleoylethanolamide, andN-arachidonoylethanolamide; in some particular embodiments, X₁ isN-palmitoylethanolamide. In some embodiments, X₂ comprises a moietyselected from the group consisting of phosphate, butyric acid, glycerol,succinate, capryhc acid, gluconoic acid, eicosapentaeonoic acid,linoleic acid, succinate, and sucrose moieties.

In some embodiments, a provided compound has a chemical structurerepresented by formula I:

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   each R¹, R², or R³ is independently hydrogen or -T-R⁴, wherein        at least one of R¹, R², or R³ is -T-R⁴;    -   -T- represents a bivalent moiety; and    -   R⁴ is an optionally substituted group selected from the group        consisting of C₁₋₄₀ aliphatic, —C(O)R, and X1; wherein        -   R is selected from the group consisting of hydrogen and            optionally substituted        -   C₁₋₂₀ aliphatic; and        -   X₁ is as defined above.

In some embodiments, a provided compound has a chemical structurerepresented by formula I-b:

X₁-X3   I-b

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   X₁ is as defined above;    -   X₃ is an optionally substituted group selected from the group        consisting of —(CH₂)_(m)—P(O)(OR)₂, C₁₋₄₀ aliphatic, -T-X₄;        further wherein        -   m is an integer select from the group consisting of 0-10;        -   -T- is as defined above;        -   X₄ is a saccharide moiety, in some particular embodiments,            X₄ is a disaccharide, for example, sucrose.

In some embodiments, at least one of R¹, R², or R³ is -T-R⁴. In someembodiments, at least two of R¹, R², or R³ is -T-R⁴.

In some embodiments, one of R¹, R², or R³ is -T-R⁴. In some embodiments,two of R¹, R², R³ are each independently -T-R⁴. In some embodiments, R¹,R², and R³ are each independently -T-R⁴.

In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is -T-R⁴.In some embodiments, R² is hydrogen. In some embodiments, R² is -T-R⁴.In some embodiments, R³ is hydrogen. In some embodiments, R³ is -T-R⁴.

In some embodiments, R¹ and R² are hydrogen, and R³ is -T-R⁴. In someembodiments, R² and R³ are hydrogen, and R¹ is -T-R⁴. In someembodiments, R¹ and R³ are hydrogen, and R² is -T-R⁴. In someembodiments, R¹ is hydrogen, and R² and R³ are each independently -T-R⁴.In some embodiments, R² is hydrogen, and R¹ and R³ are eachindependently -T-R⁴. In some embodiments. R³ is hydrogen, and R¹ and R²are each independently -T-R⁴. In some embodiments, each of R¹, R², andR³ are independently -T-R⁴.

In some embodiments, -T- represents a bivalent moiety.

In some embodiments, -T- is a bivalent moiety derived from adicarboxylic acid. In some embodiments, -T- is a bivalent moiety derivedfrom an optionally substituted dicarboxylic acid selected from the groupconsisting of oxalic acid, malonic acid, succinic acid, glutaric acid,adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid,maleic acid, fumaric acid, glutaconic acid, traumatic acid, muconicacid, glutinic acid, citraconic acid, mesaconic acid, malic acid,aspartic acid, glutamic acid, tartronic acid, tartaric acid,diaminopimelic acid, saccharic acid, mesoxalic acid, oxaloacetic acid,acetonedicarboxylic acid, arabinaric acid, phthalic acid, isophthalicacid, terephthalic acid, diphenic acid, and 2,6-napthalenedicarboxylicacid.

In some embodiments, -T- is a bivalent moiety derived from optionallysubstituted oxalic acid. In some embodiments, -T- is a bivalent moietyderived from optionally substituted malonic acid. In some embodiments,-T- is a bivalent moiety derived from optionally substituted succinicacid. In some embodiments, -T- is a bivalent moiety derived fromoptionally substituted glutaric acid. In some embodiments, -T- is abivalent moiety derived from optionally substituted adipic acid. In someembodiments, -T- is a bivalent moiety derived from optionallysubstituted pimelic acid. In some embodiments, -T- is a bivalent moietyderived from optionally substituted suberic acid. In some embodiments,-T- is a bivalent moiety derived from optionally substituted azelaicacid. In some embodiments, -T- is a bivalent moiety derived fromoptionally substituted sebacic acid. In some embodiments, -T- is abivalent moiety derived from optionally substituted maleic acid. In someembodiments, -T- is a bivalent moiety derived from optionallysubstituted fumaric acid. In some embodiments, -T- is a bivalent moietyderived from optionally substituted glutaconic acid. In someembodiments, -T- is a bivalent moiety derived from optionallysubstituted traumatic acid. In some embodiments, -T- is a bivalentmoiety derived from optionally substituted muconic acid. In someembodiments, -T- is a bivalent moiety derived from optionallysubstituted glutinic acid. In some embodiments, -T- is a bivalent moietyderived from optionally substituted citraconic acid. In someembodiments, -T- is a bivalent moiety derived from optionallysubstituted mesaconic acid. In some embodiments, -T- is a bivalentmoiety derived from optionally substituted malic acid. In someembodiments, -T- is a bivalent moiety derived from optionallysubstitutedaspartic acid. In some embodiments, -T- is a bivalent moietyderived from optionally substituted glutamic acid. In some embodiments,-T- is a bivalent moiety derived from optionally substituted tartromcacid. In some embodiments, -T- is a bivalent moiety derived fromoptionally substituted tartaric acid. In some embodiments, -T- is abivalent moiety derived from optionally substituted diaminopimelic acid.In some embodiments, -T- is a bivalent moiety derived from optionallysubstituted saccharic acid. In some embodiments, -T- is a bivalentmoiety derived from optionally substituted mesoxalic acid. In someembodiments, -T- is a bivalent moiety derived from optionallysubstituted oxaloacetic acid. In some embodiments, -T- is a bivalentmoiety derived from optionally substituted acetonedicarboxylic acid. Insome embodiments, -T- is a bivalent moiety derived from optionallysubstituted arabinaric acid. In some embodiments, -T- is a bivalentmoiety derived from optionally substituted phthalic acid. In someembodiments, -T- is a bivalent moiety derived from optionallysubstituted isophthalic acid. In some embodiments, -T- is a bivalentmoiety⁷ derived from optionally substituted terephthalic acid. In someembodiments, -T- is a bivalent moiety derived from optionallysubstituted diphenic acid. In some embodiments, -T- is a bivalentmoiety⁷ derived from optionally substituted 2,6-naphthalenedicarboxylicacid.

In some embodiments, -T-R⁴ is:

-   -   wherein R⁴ is as defined above; and    -   Y is a bivalent C₁₋₂₀ straight or branched hydrocarbon chain.

In some embodiments Y is a bivalent C₁₋₂₀ straight or branchedhydrocarbon chain. In some embodiments Y is a bivalent C₁₋₁₅ straight orbranched hydrocarbon chain. In some embodiments Y is a bivalent C₁₋₁₂straight or branched hydrocarbon chain. In some embodiments Y is abivalent C₁₋₁₀ straight or branched hydrocarbon chain. In someembodiments Y is a bivalent C₁₋₈ straight or branched hydrocarbon chain.In some embodiments Y is a bivalent C₁₋₆ straight or branchedhydrocarbon chain. In some embodiments Y is a bivalent C₁₋₅ straight orbranched hydrocarbon chain. In some embodiments Y is a bivalent C₁₋₄straight or branched hydrocarbon chain. In some embodiments Y is abivalent C₁₋₃ straight or branched hydrocarbon chain. In someembodiments Y is a bivalent C₁₋₂ straight or branched hydrocarbon chain.

In some embodiments Y is a bivalent C₁₋₂₀ straight hydrocarbon chain. Insome embodiments Y is a bivalent C₁₋₁₅ straight hydrocarbon chain. Insome embodiments Y is a bivalent C₁₋₁₂ straight hydrocarbon chain. Insome embodiments Y is a bivalent C₁₋₄ straight hydrocarbon chain. Insome embodiments Y is a bivalent C₁₋₈ straight hydrocarbon chain. Insome embodiments Y is a bivalent C₁₋₆ straight hydrocarbon chain. Insome embodiments Y is a bivalent C₁₋₅ straight hydrocarbon chain. Insome embodiments Y is a bivalent C₁₋₄ straight hydrocarbon chain. Insome embodiments Y is a bivalent C₁₋₃ straight hydrocarbon chain. Insome embodiments Y is a bivalent C₁₋₂ straight hydrocarbon chain.

In some embodiments Y is a bivalent C₁ hydrocarbon chain. In someembodiments Y is a bivalent C₂ straight or branched hydrocarbon chain.In some embodiments Y is a bivalent C₃ straight or branched hydrocarbonchain. In some embodiments Y is a bivalent C₄ straight or branchedhydrocarbon chain. In some embodiments Y is a bivalent C₅ straight orbranched hydrocarbon chain. In some embodiments Y is a bivalent C₆,straight or branched hydrocarbon chain. In some embodiments Y is abivalent C₁₀ straight or branched hydrocarbon chain.

In some embodiments, Y is propylene. In some embodiments, Y is ethylene.In some embodiments, Y is methylene.

In some embodiments, at least one of R¹, R², or R³ is

In some embodiments, at least two of R¹, R², or R³ are eachindependently

In some embodiments, one of R¹, R², or R³ is

In some embodiments, R¹ is

In some embodiments, R² is

In some embodiments, two of R¹, R², or R³ are each independently

In some embodiments, R¹, P², and R³ are each independently

In some embodiments, R⁴ is an optionally substituted group selected fromthe group consisting of C₁₋₄₀ aliphatic, —C(O)R, and X₁. In someembodiments, R⁴ is optionally substituted C₁₋₄₀ aliphatic. In someembodiments, R⁴ is optionally substituted C₁₋₃₅ aliphatic. In someembodiments, R⁴ is optionally substituted C₁₋₃₀ aliphatic. In someembodiments, R⁴ is optionally substituted C₁₋₂₅ aliphatic. In someembodiments, R⁴ is optionally substituted C₁₋₂₀ aliphatic, someembodiments, R⁴ is optionally substituted C₁₋₁₀ aliphatic. In someembodiments R⁴ is optionally substituted C₁₋₆ aliphatic. In someembodiments, R⁴ is optionally substituted —C(O)R. In some embodiments,R⁴ is X₁.

In some embodiments, X₁ is selected from N-palmitoylethanolamide,N-oleoylethanolamide, or N-arachidonoylethanolamide. In someembodiments, X₁ is N-palmitoylethanolamide. In some embodiments, X₁ isN-oleoylethanolamide. In some embodiments, X₁ isN-arachidonoylethanolamide.

In some embodiments, R is selected from the group consisting of hydrogenand optionally substituted C₁₋₂₀ aliphatic. In some embodiments. R ishydrogen. In some embodiments, R is optionally substituted C₁₋₂₀aliphatic. In some embodiments, R is optionally substituted C₁₋₁₀aliphatic. In some embodiments, R is optionally substituted C₁₋₆aliphatic. In some embodiments, R is optionally substituted C₁₋₃aliphatic.

In some embodiments, X₃ is an optionally substituted group selected fromthe group consisting of —(CH₂)_(m)—P(O)(OR)₂, C₁₋₄₀ aliphatic, and-T-X₄. In some embodiments, X₃ is optionally substituted—(CH₂)_(m)—P(O)(OR)₂. In some embodiments, X₃ is optionally substitutedC₁₋₄₀ aliphatic. In some embodiments, X₃ is optionally substituted C₁₋₃₀aliphatic. In some embodiments, X₃ is optionally substituted C₁₋₂₀aliphatic. In some embodiments. X₃ is optionally substituted C₁₋₁₀aliphatic. In some embodiments, X₃ is optionally substituted C₁₋₆aliphatic. In some embodiments, X₃ is an optionally substituted -T-X₄.

In some embodiments, in is an integer select from the group consistingof 0-10. In some embodiments, m is an integer select from the groupconsisting of 0-5. In some embodiments, m is 0. In some embodiments, inis 1. In some embodiments, in is 2. In some embodiments, m is 3. In someembodiments, in is 4. In some embodiments, m is 5.

In some embodiments, X₄ is a saccharide moiety. In some embodiments, X₄is a disaccharide. In some embodiments, X₄ is sucrose.

In some embodiments, a compound of formula I does not comprise astereocenter within the glycerol backbone (e.g., when R¹ and R³ are thesame). In some embodiments, a compound of formula I comprises astereocenter within the glycerol backbone (e.g., wherein R¹ and R³ aredifferent). In some embodiments, a compound of formula I is providedand/or utilized as a racemic mixture. In some embodiments, a compound offormula I is provided and/or utilized as a mixture of stereoforms thatmay or may not be a racemic mixture. In some embodiments, a compound offormula I is provided and/or utilized as a single enantiomer. In someembodiments, the present disclosure provides compounds of formula I′ orI″:

-   -   or a pharmaceutically acceptable salt thereof;    -   wherein R¹, R², and R³ are as defined above.

The present disclosure also provides the insight that, in someembodiments, the position of the N-acylethanolamide, e.g., PEA, on theglycerol moiety may have an effect on its pharmacological properties.For example, a glycerol moiety with an N-acylethanolamide moietyconjugated to the 2 position (e.g., the position corresponding to *—OR²of formulae I, I′, or I″) may exhibit improved pharmacologicalproperties over a glycerol moiety with an N-acylethanolamide moietyconjugated to the 1 or 3 position (e.g., the position corresponding to*—OR¹ or *—OR³ of formulae I, I′, or I″). Without wishing to be bound toa particular theory, the present disclosure proposes that, in someembodiments, the 1 and 3 positions of the glycerol backbone may be moresusceptible to cellular lipases than the 2 position.

The present disclosure also provides the insight that, in someembodiments, a compound provided herein may isomerize, for example,undergoing positional isomerization. For example, the present disclosureproposes that, in some embodiments, when a glycerol moiety comprises afree alcohol (e.g., a free alcohol at a position corresponding to *—OR¹or *—OR³ of formulae I, I′, or I″), a moiety conjugated to glycerol(e.g., a moiety comprising an N-acylethanolamide at a positioncorresponding to *—OR² of formulae I, I′, or I″), may migrate to a freealcohol (e.g., migrate from a position corresponding to *—OR² offormulae I, I′, or I″ to a position corresponding to *—OR¹ or *—OR³ offormulae I, I′, or I″). For example, compounds I-8 and I-9 mayinterconvert among positional isomers.

In some embodiments, isomerization occurs prior to administration. Insome embodiments, isomerization occurs after administration.

In addition, the present disclosure provides the insight that, in someembodiments, a glycerol moiety that does not comprise a free alcoholwill not isomerize. For example, in some embodiments, compound I-16 doesnot undergo positional isomerization.

In some embodiments, the present disclosure provides compounds offormula II:

or a pharmaceutically acceptable salt thereof;wherein Y and R⁴ are as defined above.

In some embodiments, the present disclosure provides compounds offormula III:

or a pharmaceutically acceptable salt thereof;wherein Y and R⁴ are as defined above.

In some embodiments, the present disclosure provides compounds offormulae III′ or III″

or a pharmaceutically acceptable salt thereof;wherein Y and R⁴ are as defined above.

In some embodiments, the present disclosure provides compounds offormula IV:

or a pharmaceutically acceptable salt thereof;wherein:

-   -   R^(4a) and R^(4b) are independently hydrogen, —C(O)R′, or        —C(O)—Y—C(O)OR′;        -   wherein        -   each R^(:) is independently selected from the group            consisting of hydrogen and an optionally substituted C₁₋₂₀            aliphatic; and        -   each Y is independently as defined above and described            herein.

In some embodiments, the present disclosure provides compounds offormulae IV′ or IV″:

or a pharmaceutically acceptable salt thereof; wherein R^(4a) and R^(4b)are as defined above and herein.

In some embodiments, the present disclosure provides compounds offormula V:

wherein:

-   -   R^(4a) and R^(4c) are independently hydrogen, —C(O)R′, or        —C(O)—Y—C(O)OR′;    -   wherein        -   each R′ is independently selected from the group consisting            of hydrogen and an optionally substituted C₁₋₂₀ aliphatic;            and        -   each Y is independently as defined above and described            herein.

In some embodiments, the present disclosure provides compounds offormulae V′ or V″

or a pharmaceutically acceptable salt thereof;wherein R^(4a) and R^(4c) are as defined above and herein.

In some embodiments, R^(4a) is hydrogen. In some embodiments, R^(4a) is—C(O)R′. In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′. In someembodiments, R^(4b) is hydrogen. In some embodiments, R^(4b) is —C(O)R′.In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′. In some embodiments.R^(4c) is hydrogen. In some embodiments, R^(4c) is —C(O)R′. In someembodiments, R^(4c) is is —C(O)—Y—C(O)OR′.

In some embodiments, R′ is hydrogen. In some embodiments, R′ isoptionally substituted C₁₋₂₀ aliphatic. In some embodiments. R′ isselected from the group consisting of: hydrogen,

In some embodiments, R′ is selected from the group consisting of:

In some embodiments, R′ is

In some embodiments, R′ is

In some embodiments, R′ is

In some embodiments, R′ is

In some embodiments R′ is

In some embodiments, R^(4a) is —C(O)R′. In some embodiments, R^(4a) is—C(O)R′, wherein R′ is optionally substituted C₁₋₂₀ aliphatic. In someembodiments, R^(4a) is selected from the group consisting of:

In some embodiments, R^(4a) is selected from the group consisting of:

In some embodiments, R^(4a) is

In some embodiments, R^(4a) is

In some embodiments, R^(4a) is

In some embodiments, R^(4a) is

In some embodiments, R^(4a) is

In some embodiments, R^(4a) is —C(O)—Y—C(O)OR″. In some embodiments,R^(4a) is —C(O)—Y—C(O)OR″, wherein R′ is optionally substituted C₁₋₂₀aliphatic.

In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein R′ is selectedfrom the group consisting of hydrogen,

In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein R′ is hydrogen.In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein R′ is selectedfrom the group consisting of

In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁₋₂₀ straight or branched hydrocarbon chain. In some embodiments,R^(4a) is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₁₅ straight orbranched hydrocarbon chain. In some embodiments, R^(4a) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₁₂ straight or branchedhydrocarbon chain. In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₁₋₁₀ straight or branched hydrocarbon chain. Insome embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁₋₈ straight or branched hydrocarbon chain. In some embodiments, R^(4a)is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₆ straight or branchedhydrocarbon chain. In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₁₋₅ straight or branched hydrocarbon chain. Insome embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁₋₄ straight or branched hydrocarbon chain. In some embodiments, R^(4a)is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₃ straight or branchedhydrocarbon chain. In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₁₋₂ straight or branched hydrocarbon chain.

In some embodiments, R^(4a) is —C(O)—Y—C(O)OR, wherein Y is a bivalentC₁₋₂₀ straight hydrocarbon chain. In some embodiments, R^(4a) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₁₅ straight hydrocarbonchain. In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein Y is abivalent C₁₋₁₂ straight hydrocarbon chain. In some embodiments, R^(4a)is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₁₀ straight hydrocarbonchain. In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein Y is abivalent C₁₋₈ straight hydrocarbon chain. In some embodiments, R^(4a) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₆ straight hydrocarbonchain. In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein Y is abivalent C₁₋₅ straight hydrocarbon chain. In some embodiments, R^(4a) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₄ straight hydrocarbonchain. In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′ wherein Y is abivalent C₁₋₃ straight hydrocarbon chain. In some embodiments, R^(4a) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₂ straight hydrocarbonchain.

In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁ hydrocarbon chain. In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₂ straight or branched hydrocarbon chain. Insome embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₃straight or branched hydrocarbon chain. In some embodiments, R^(4a) is—C(O)—Y—C(O)OR, wherein Y is a bivalent C₄ straight or branchedhydrocarbon chain. In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₅ straight or branched hydrocarbon chain. Insome embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₆straight or branched hydrocarbon chain. In some embodiments, R^(4a) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₀ straight or branchedhydrocarbon chain.

In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein, Y is propylene.In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein Y is ethylene.In some embodiments, R^(4a) is —C(O)—Y—C(O)OR′, wherein Y is methylene.

In some embodiments, R^(4a) is

In some embodiments, R^(4b) is —C(O)R′ In some embodiments, R^(4b) is—C(O)R′, wherein R′ is optionally substituted C₁₋₂₀ aliphatic. In someembodiments, R^(4b) is selected from the group consisting of:

In some embodiments, R^(4b) is selected from the group consisting of:

In some embodiments, R^(4b) is

In some embodiments, R^(4b) is

In some embodiments, R^(4b) is

In some embodiments, R^(4b) is

In some embodiments, R^(4b) is

In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′. In some embodiments,R^(4b) is —C(O)—Y—C(O)OR′, wherein R′ is optionally substituted C₁₋₂₀aliphatic.

In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′, C(O)OR′, wherein R′ isselected from the group consisting of,

In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein R′ is hydrogen.In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein R′ is selectedfrom the group consisting of

In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁₋₂₀ straight or branched hydrocarbon chain. In some embodiments,R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₁₅ straight orbranched hydrocarbon chain. In some embodiments, R^(4b) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₁₂ straight or branchedhydrocarbon chain. In some embodiments, R is —C(O)—Y—C(O)OR′, wherein Yis a bivalent C₁₋₁₀ straight or branched hydrocarbon chain. In someembodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₈straight or branched hydrocarbon chain. In some embodiments, R^(4b) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₆ straight or branchedhydrocarbon chain. In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₁₋₅ straight or branched hydrocarbon chain. Insome embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁₋₄ straight or branched hydrocarbon chain. In some embodiments, R^(4b)is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₃ straight or branchedhydrocarbon chain. In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₁₋₂ straight or branched hydrocarbon chain.

In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁₋₂₀ straight hydrocarbon chain. In some embodiments, R^(4b) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₁₅ straight hydrocarbonchain. In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is abivalent C₁₋₁₂ straight hydrocarbon chain. In some embodiments, R^(4b)is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₁₀ straight hydrocarbonchain. In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is abivalent C₁₋₈ straight hydrocarbon chain. In some embodiments, R^(4b) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₆ straight hydrocarbonchain. In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is abivalent C₁₋₅ straight hydrocarbon chain. In some embodiments, R^(4b) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₄ straight hydrocarbonchain. In some embodiments, R^(4b) is —C(O)—Y—C(O)OR, wherein Y is abivalent C₁₋₃ straight hydrocarbon chain. In some embodiments. R^(4b) is—C(O)—Y—C(O)OR, wherein Y is a bivalent C₁₋₂ straight hydrocarbon chain.

In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁ hydrocarbon chain. In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₂ straight or branched hydrocarbon chain. Insome embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₃straight or branched hydrocarbon chain. In some embodiments, R^(4b) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₄ straight or branchedhydrocarbon chain. In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₅ straight or branched hydrocarbon chain. Insome embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₆straight or branched hydrocarbon chain. In some embodiments, R^(4b) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₀ straight or branchedhydrocarbon chain.

In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein, Y is propylene.In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is ethylene.In some embodiments, R^(4b) is —C(O)—Y—C(O)OR′, wherein Y is methylene.

In some embodiments, R^(4b) is

In some embodiments, R^(4c) is —C(O)R′. In some embodiments, R^(4c) is—C(O)R′, wherein R′ is optionally substituted C₁₋₂₀ aliphatic. In someembodiments, R^(4c) is selected from the group consisting of:

In some embodiments, R^(4c) is selected from the group consisting of:

In some embodiments R^(4c) is

In some embodiments R^(4c) is

In some embodiments R^(4c) is

In some embodiments R^(4c) is

In some embodiments R^(4c) is

In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′. In some embodiments,R^(4c) is —C(O)—Y—C(O)OR′, wherein R′ is optionally substituted C₁₋₂₀aliphatic.

In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein R′ is selectedfrom the group consisting of hydrogen,

In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein R′ is hydrogen.In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein R′ is selectedfrom the group consisting of

In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁₋₂₀ straight or branched hydrocarbon chain. In some embodiments,R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₁₅ straight orbranched hydrocarbon chain. In some embodiments, R^(4c) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₁₂ straight or branchedhydrocarbon chain. In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₁₋₁₀ straight or branched hydrocarbon chain. Insome embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁₋₈ straight or branched hydrocarbon chain. In some embodiments, R^(4c)is —C(O)—Y—C(O)OR′, is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₅straight or branched hydrocarbon chain. In some embodiments. R^(4c) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₄ straight or branchedhydrocarbon chain. In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₁₋₃ straight or branched hydrocarbon chain. Insome embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁₋₂ straight or branched hydrocarbon chain.

In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁₋₂₀ straight hydrocarbon chain. In some embodiments, R^(4c) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₁₅ straight hydrocarbonchain. In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is abivalent C₁₋₁₂ straight hydrocarbon chain. In some embodiments, R^(4c)is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₁₀ straight hydrocarbonchain. In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is abivalent C₁₋₈ straight hydrocarbon chain. In some embodiments, R^(4c) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₆ straight hydrocarbonchain. In some embodiments, R⁴ is —C(O)—Y—C(O)OR′, wherein Y is abivalent C₁₋₅ straight hydrocarbon chain. In some embodiments, R^(4c) is—C(O)—Y—C(O)O—R′, wherein Y is a bivalent C₁₋₄ straight hydrocarbonchain. In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is abivalent C₁₋₃ straight hydrocarbon chain. In some embodiments, R^(4c) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₋₂ straight hydrocarbonchain.

In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is a bivalentC₁ hydrocarbon chain. In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₂ straight or branched hydrocarbon chain. Insome embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₃straight or branched hydrocarbon chain. In some embodiments, R^(4c) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₄ straight or branchedhydrocarbon chain. In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′,wherein Y is a bivalent C₅ straight or branched hydrocarbon chain. Insome embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is a bivalent C₆straight or branched hydrocarbon chain. In some embodiments, R^(4c) is—C(O)—Y—C(O)OR′, wherein Y is a bivalent C₁₀ straight or branchedhydrocarbon chain.

In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein, Y is propylene.In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is ethylene.In some embodiments, R^(4c) is —C(O)—Y—C(O)OR′, wherein Y is methylene.

In some embodiments, R^(4c) is

In some embodiments, each of R^(4a), R^(4b), and R^(4c) is hydrogen. Insome embodiments, each of R^(4a), R^(4b), and R^(4c) is independentlyselected from the group consisting of:

In some embodiments, each of R^(4a), R^(4b), and R^(4c) is independentlyselected from the group consisting of:

In some embodiments, each of R^(4a), R^(4b), and R^(4c) is independentlyselected from the group consisting of:

In some embodiments, R^(4a) and R^(4b) or R^(4a) and R^(4c) are thesame. In some embodiments, R^(4a) and R^(4b) are the same. In someembodiments, R^(4a) and R^(4b) are

In some embodiments, R^(4a) and R^(4b) are

In some embodiments, R^(4a) and R^(4b) are

In some embodiments, R^(4a) and R^(4b) are

In some embodiments, R^(4a) and R^(4b) are

In some embodiments, R^(4a) and R^(4b) are

In some embodiments, R^(4a) and R^(4b) are

In some embodiments, R^(4a) and R^(4c) are the same. In someembodiments, R^(4a) and R^(4c) are

In some embodiments, R^(4a) and R^(4c) are

In some embodiments, R^(4a) and R^(4c) are

In some embodiments, R^(4a) and R^(4c) are

some embodiments, and R^(4c) are

In some embodiments, R^(4a) and R^(4c) are

In some embodiments, R^(4a) and R^(4c) are

In some embodiments, the present disclosure provides N-acylethanolamidederivatives selected from those in Table 1.

TABLE 1

I-a-2

I-a-3

I-a-5

I-a-6

I-1

I-2

I-3

I-4

I-5

I-6

I-7

I-8

I-9

In some embodiments, the present disclosure provides compounds selectedfrom those in Table 1-a.

TABLE 1-a

I-10

I-11

I-12

I-13

I-14

I-15

I-16

In some embodiments, one or more hydrogen atoms are replaced with adeuterium atom(s). In some embodiments, one or more of R¹, R², or R³ isor contains deuterium.

In some embodiments, provided N-acylethanolamide derivative compoundsdisplay one or more activities that is/are comparable to that of areference compound (e.g., a parent N-acylethanolamide compound). In someembodiments, provided N-acylethanolamide derivative compounds displayone or more activities that is/are improved as compared to a referencecompound (e.g., a parent N-acylethanolamide compound).

In some embodiments, provided N-acylethanolamide derivative compoundsare characterized in that the compound may show improved solubility inan aqueous system as compared to a reference compound (e.g., a parentN-acylethanolamide compound). In some particular embodiments, aqueoussolubility may be assessed according to an appropriate assay. In someembodiments, an appropriate assay is known in the art and/or describedherein.

In some embodiments, provided N-acylethanolamide derivative compoundsare characterized in that the compound may show improved stability ascompared to a reference compound (e.g., a parent N-acylethanolamidecompound) In some particular embodiments, stability may be assessed, forexample, using an appropriate assay. In some embodiments, an appropriateassay is known in the art and/or described herein.

In some embodiments, provided N-acylethanolamide derivative compoundsare characterized in that the compound is metabolized differently ascompared to a reference compound (e.g., a parent N-acylethanolamidecompound). In some embodiments, provided N-acylethanolamide derivativecompounds are characterized in that the compound is metabolized at adifferent rate as compared to a reference compound (e.g., a parentN-acylethanolamide compound). In some embodiments, providedN-acylethanolamide derivative compounds are characterized in that thecompound is metabolized at a faster rate as compared to a referencecompound (e.g., a parent N-acylethanolamide compound). In someembodiments, provided N-acylethanolamide derivative compounds arecharacterized in that the compound is metabolized at a slower rate ascompared to a reference compound (e.g., a parent N-acylethanolamidecompound). In some particular embodiments, metabolized rate may beassessed, for example, using an appropriate assay. In some embodiments,an appropriate assay is known in the art and/or described herein.

In some embodiments, provided N-acylethanolamide derivative compoundsare characterized in that when administered, the compound delivers aparent N-acylethanolamide compound or an active metabolite thereof.

In some embodiments, provided N-acylethanolamide derivative compoundsare characterized in that when administered, the compound exhibits animproved oral bioavailability as compared to a reference compound (e.g.,a parent N-acylethanolamide compound). In some particular embodiments,oral bioavailability may be assessed, for example, using an appropriateassay. In some embodiments, an appropriate assay is known in the artand/or described herein.

In some embodiments, a reference compound is or comprises a parentN-acylethanolamide compound. In some embodiments, a reference compoundis or comprises palmitoylethanolamide.

In some embodiments, provided N-acylethanolamide derivative compoundsdisplay one or more activities that is/are comparable to that ofpalmitoylethanolamide. In some embodiments, provided N-acylethanolamidederivative compounds display one or more activities that is/are improvedas compared to palmitoylethanolamide.

In some embodiments, provided N-acylethanolamide derivative compoundsdisplay increased solubility as compared to that ofpalmitoylethanolamide. In some particular embodiments, aqueoussolubility may be assessed, for example, using an appropriate assay. Insome embodiments, an appropriate assay is known in the art and ordescribed herein.

In some embodiments, provided N-acylethanolamide derivative compoundsdisplay increased stability that is comparable to that ofpalmitoylethanolamide.

In some embodiments, provided N-acylethanolamide derivative compoundsare metabolized differently as compared to that ofpalmitoylethanolamide. In some embodiments, provided N-acylethanolamidederivative compounds are metabolized at a faster rate as compared tothat of palmitoylethanolamide. In some embodiments, providedN-acylethanolamide derivative compounds are metabolized at a slower rateas compared to that of palmitoylethanolamide.

In some embodiments, provided N-acylethanolamide derivative compoundswhen administered deliver palmitoylethanolamide or an active metabolitethereof. In some embodiments, provided N-acylethanolamide derivativecompounds when administered display improved oral bioavailability ascompared to the administration of palmitoylethanolamide.

In some embodiments, a reference compound is or comprisesoleoylethanolamide.

In some embodiments, provided N-acylethanolamide derivative compoundsdisplay one or more activities that is/are comparable to that ofoleoylethanolamide. In some embodiments, provided N-acylethanolamidederivative compounds display one or more activities that is/are improvedas compared to oleoylethanolamide.

In some embodiments, provided N-acylethanolamide derivative compoundsdisplay increased solubility that is comparable to that ofoleoylethanolamide. In some particular embodiments, aqueous solubilitymay be assessed, for example, using an appropriate assay. In someembodiments, an appropriate assay is known in the art and or describedherein.

In some embodiments, provided N-acylethanolamide derivative compoundsdisplay increased stability that is comparable to that ofoleoylethanolamide.

In some embodiments, provided N-acylethanolamide derivative compoundsare metabolized differently as compared to that of oleoylethanolamide.In some embodiments, provided N-acylethanolamide derivative compoundsare metabolized at a faster rate as compared to that ofoleoylethanolamide. In some embodiments, provided N-acylethanolamidederivative compounds are metabolized at a slower rate as compared tothat of oleoylethanolamide.

In some embodiments, provided N-acylethanolamide derivative compoundswhen administered deliver oleoylethanolamide or an active metabolitethereof. In some embodiments, provided N-acylethanolamide derivativecompounds when administered display improved oral bioavailability ascompared to the administration of oleoylethanolamide.

In some embodiments, a reference compound is or comprisesarachidonylethanolamide.

In some embodiments, provided N-acylethanolamide derivative compoundsdisplay one or more activities that is/are comparable to that ofarachidonylethanolamide. In some embodiments, providedN-acylethanolamide derivative compounds display one or more activitiesthat is/are improved as compared to arachidonylethanolamide.

In some embodiments, provided N-acylethanolamide derivative compoundsdisplay increased solubility that is comparable to that ofarachidonylethanolamide. In some particular embodiments, aqueoussolubility may be assessed, for example, using an appropriate assay. Insome embodiments, an appropriate assay is known in the art and/ordescribed herein.

In some embodiments, provided N-acylethanolamide derivative compoundsdisplay increased stability that is comparable to that ofarachidonylethanolamide.

In some embodiments, provided N-acylethanolamide derivative compoundsare metabolized differently as compared to that ofarachidonylethanolamide. In some embodiments, providedN-acylethanolamide derivative compounds are metabolized at a faster rateas compared to that of arachidonylethanolamide. In some embodiments,provided N-acylethanolamide derivative compounds are metabolized at aslower rate as compared to that of arachidonylethanolamide.

In some embodiments, provided N-acylethanolamide derivative compoundswhen administered deliver arachidonylethanolamide or an activemetabolite thereof. In some embodiments, provided N-acylethanolamidederivative compounds when administered display improved oralbioavailability as compared to the administration ofarachidonylethanolamide.

Uses

In some embodiments, the present disclosure provides methods ofidentifying and/or characterizing derivatives of an N-acylethanolamidecompound (e.g., a parent N-acylethanolamide compound), which methodcomprising the steps of:

-   -   providing a derivative compound comprising a moiety modifying or        otherwise linked to an N-acylethanolamide; and    -   determining that the derivative compound has one or more        improved pharmacologic properties relative to the        N-acylethanolamide compound.

In some embodiments, the present disclosure provides technologies foridentifying, assessing, and/or characterizing one or more activities orattributes of one or more provided N-acylethanolamide derivativecompounds.

In some embodiments, the present disclosure provides methods of treatinga subject suffering from or susceptible to a disease, disorder, orcondition, which method comprises a step of:

-   -   administering an N-acylethanolamide derivative or composition        disclosed herein to a subject in need thereof.

In some embodiments, an N-acylethanolamide derivative or compositiondisclosed herein is administered in combination with one or more otheragents that treat the relevant disease, disorders, or conditions (or oneor more symptoms thereof) from which a relevant subject is suffering.

Various diseases, disorders, and/or conditions may be affected by anN-acylethanolamide. In some embodiments, the present disclosure providesmethods comprising administering to a subject suffering from orsusceptible to a disease, disorder, or condition a pharmaceuticallyeffective amount of a provided compound or composition.

In some embodiments, a disease, disorder, or condition is or comprisespain. In some embodiments, pain may be chronic pain. In someembodiments, pain may be or include lower back pain. In someembodiments, a disease, disorder, or condition is or comprises chroniclower back pain. In some embodiments, a disease, disorder, or conditionis or comprises sciatica. In some embodiments, a disease, disorder, orcondition is or comprises radiculopathy. In some embodiments, a disease,disorder, or condition is or comprises radiating pain. Certain painclassification and representative indications are depicted in FIG. 1.

In some embodiments, a disease, disorder, or condition is or comprisesanxiety. In some embodiments, a disease, disorder, or condition is orcomprises depression. In some embodiments, a disease, disorder, orcondition is characterized by one or more symptoms of schizophrenia.

In some embodiments, a disease, disorder, or condition is or comprises aneurologic, disease, disorder, or condition. In some embodiments, adisease, disorder or condition is or comprises Huntington's disease. Insome embodiments, a disease, disorder or condition is or comprisesParkinson's disease. In some embodiments, a disease, disorder orcondition is or comprises Alzheimer's disease. In some embodiments, adisease, disorder, or condition is or comprises Amyotrophic LateralSclerosis (ALS, also known as Lou Gehrig's disease). In someembodiments, a disease, disorder, or condition is or comprises multiplesclerosis. In some embodiments, a disease, disorder, or condition is orcomprises neuropathic pain. In some embodiments, a disease, disorder, orcondition is or comprises cerebral ischemia. In some embodiments, adisease, disorder, or condition is or comprises epilepsy.

In some embodiments, a disease, disorder, or condition is or comprisesappetite loss. In some embodiments, a disease, disorder, or condition isor comprises dental pain. In some embodiments, a disease, disorder, orcondition is or comprises osteoarthritis. In some embodiments, adisease, disorder, or condition is or comprises reduced gastrointestinalmotility.

In some embodiments, a disease, disorder, or condition is or comprisescancer.

In some embodiments, a disease, disorder, or condition is or comprisesan ophthalmic condition. In some embodiments, a disease, disorder, orcondition is or comprises glaucoma.

In some embodiments, a disease, disorder, or condition is or comprisesatopic dermatitis. In some embodiments, a disease, disorder, orcondition is or comprises respiratory infection. In some embodiments, adisease, disorder, or condition is or comprises post-traumatic stressdisorder. In some embodiments, a disease, disorder, or condition is orcomprises obesity. In some embodiments, a disease, disorder, orcondition is or comprises insomnia. In some embodiments, a disease,disorder, or condition is or comprises sleepiness.

In some embodiments, the present disclosure provides methods of reducinggastrointestinal motility in a patient, which method comprising the stepof administering a compound or composition disclosed herein to a subjectin need thereof.

In some embodiments, the present disclosure provides methods of reducingcancer cell proliferation in a patient or in a biological sample, whichmethod comprising the step of administering to said patient orcontacting said biological sample with a compound or compositiondisclosed herein.

In some embodiments, the present disclosure provides methods of inducinglipolysis in a patient or in a biological sample, which methodcomprising the step of administering to said patient or contacting saidbiological sample with a compound or composition disclosed herein.

In some particular embodiments, provided compounds including a butyricacid moiety is useful in the treatment of IBS-D and/or for the treatmentof pain. Butyric acid (BA) is a critical component of gut heath and hasbeen shown to decrease pain, reduce frequency and increased consistencyof bowel movements for IBS-D patients.

In some embodiments, provided treatments that utilize one or morecompounds as described herein (e.g., treatment of pain) may deliver PEAat a level corresponding to a dose greater than or equal to 1200 mg/dayPEA. In some embodiments, provided treatments may involve administrationonce or twice daily.

In some embodiments, provided treatments that utilize one or morecompounds as described herein (e.g., treatment of IBS-D) may deliver PEAat a level corresponding to a dose of about 3 g/day PEA.

In some embodiments, provided treatments withbutyric-acid-moiety-containing compounds as described herein (e.g.,treatment of pain and/or IBS-D) may deliver PEA at a level correspondingto a dose of about 3 g/day PEA and/or may deliver BA at a levelcorresponding to a dose of about 1 g/day BA.

In some embodiments, one or more particular compounds provided hereinmay be useful in the treatment of a plurality of different diseases,disorders or conditions; om some such embodiments, the compound may bedifferently formulated when utilized for different diseases, disordersor conditions.

Compositions

In some embodiments, compounds as provided herein are prepared and/orutilized in compositions, such as pharmaceutical compositions. In someembodiments, a provided pharmaceutical composition comprises atherapeutically effective amount of a provided compound, and at leastone pharmaceutically acceptable inactive ingredient selected frompharmaceutically acceptable diluents, pharmaceutically acceptableexcipients, and pharmaceutically acceptable carriers. In someembodiments, the pharmaceutical composition is formulated forintravenous injection, oral administration, buccal administration,inhalation, nasal administration, topical administration, ophthalmicadministration or optic administration. In some embodiments, thepharmaceutical composition is a tablet, a pill, a capsule, a liquid, aninhalant, a nasal spray solution, a suppository, a suspension, a gel, acolloid, a dispersion, a suspension, a solution, an emulsion, anointment, a lotion, an eye drop or an ear drop.

In some embodiments, the present disclosure provides a pharmaceuticalcomposition comprising a provided compound or composition, in a mixturewith a pharmaceutically acceptable excipient.

In therapeutic and/or diagnostic applications, provide compounds can beformulated for a variety of modes of administration, including systemicand topical or localized administration. Techniques and formulationsgenerally may be found in Remington, The Science and Practice ofPharmacy, (20th ed. 2000).

Provided compounds and compositions thereof are effective over a widedosage range. For example, in the treatment of adult humans, dosagesfrom about 0.01 to about 10000 mg, from about 0.01 to about 1000 mg,from about 0.5 to about 100 mg, from about 1 to about 50 mg per day, andfrom about 5 to about 100 mg per day are examples of dosages that may beused. The exact dosage will depend upon the route of administration, theform in which the compound is administered, the subject to be treated,the body weight of the subject to be treated, and the preference andexperience of the attending physician.

Pharmaceutically acceptable salts are generally well known to those ofordinary skill in the art, and may include, by way of example but notlimitation, acetate, benzenesulfonate, besylate, benzoate, bicarbonate,bitartrate, bromide, calcium edetate, camsylate, carbonate, citrate,edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate, mucate,napsylate, nitrate, pamoate (embonate), pantothenate,phosphate/diphosphate, polygalacturonate, salicylate, stearate,subacetate, succinate, sulfate, tannate, tartrate, or teoclate. Otherpharmaceutically acceptable salts may be found in, for example,Remington, The Science and Practice of Pharmacy (20th ed. 2000).Preferred pharmaceutically acceptable salts include, for example,acetate, benzoate, bromide, carbonate, citrate, gluconate, hydrobromide,hydrochloride, maleate, mesylate, napsylate, pamoate (embonate),phosphate, salicylate, succinate, sulfate, or tartrate.

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained-low release form as is known to those skilled in theart. Techniques for formulation and administration may be found inRemington, The Science and Practice of Pharmacy (20th ed. 2000).Suitable routes may include oral, buccal, by inhalation spray,sublingual, rectal, transdermal, vaginal, transmucosal, nasal orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intra-articullar, intra-sternal,intra-synovial, intra-hepatic, intralesional, intracranial,intraperitoneal, intranasal, or intraocular injections or other modes ofdelivery.

For injection, provided agents may be formulated and diluted in aqueoussolutions, such as in physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiological saline buffer. For suchtransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

Use of pharmaceutically acceptable inert carriers to formulate providedcompounds or compositions into dosages suitable for systemicadministration is within the scope of the disclosure. With proper choiceof carrier and suitable manufacturing practice, the compositions of thepresent disclosure, in particular, those formulated as solutions, may beadministered parenterally, such as by intravenous injection.

The compounds can be formulated readily using pharmaceuticallyacceptable carriers well known in the art into dosages suitable for oraladministration. Such carriers enable provided compounds and compositionsto be formulated as tablets, pills, capsules, liquids, gels, syrups,slurries, suspensions and the like, for oral ingestion by a subject(e.g., patient) to be treated.

For nasal or inhalation delivery, provided compounds or compositions mayalso be formulated by methods known to those of skill in the art, andmay include, for example, but not limited to, examples of solubilizing,diluting, or dispersing substances such as, saline, preservatives, suchas benzyl alcohol, absorption promoters, and fluorocarbons.

In certain embodiments, provided compounds and compositions aredelivered to the CNS. In certain embodiments, provided compounds andcompositions are delivered to the cerebrospinal fluid. In certainembodiments, provided compounds and compositions are administered to thebrain parenchyma. In certain embodiments, provided compounds andcompositions are delivered to an animal/subject by intrathecaladministration, or intracerebroventricular administration. Broaddistribution of provided compounds and compositions, described herein,within the central nervous system may be achieved with intraparenchymaladministration, intrathecal administration, or intracerebroventricularadministration.

In certain embodiments, parenteral administration is by injection, by,e.g., a syringe, a pump, etc. In certain embodiments, the injection is abolus injection. In certain embodiments, the injection is administereddirectly to a tissue, such as striatum, caudate, cortex, hippocampus andcerebellum.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, gels, syrups, suspensions, powders, orsolutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP: povidone). If desired, disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

In some embodiments, cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,polyethylene glycol (PEG), and/or titanium dioxide, lacquer solutions,and suitable organic solvents or solvent mixtures. Dye-stuffs orpigments may be added to the tablets or dragee coatings foridentification or to characterize different combinations of activecompound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in a mixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

Depending upon the particular condition, or disease state, to be treatedor prevented, additional therapeutic agents, which are normallyadministered to treat or prevent that condition, may be administeredtogether with provided compounds or compositions. For example,chemotherapeutic agents or other anti-proliferative agents may becombined with provided compounds or compositions to treat proliferativediseases and cancer. Examples of known chemotherapeutic agents include,but are not limited to, adriamycin, dexamethasone, vincristine,cyclophosphamide, fluorouracil, topotecan, taxol, interferons, andplatinum derivatives.

Methods of Making

In some embodiments, the present disclosure provides methods ofmanufacturing a provided N-acylethanolamide derivative compound, whichmethod comprising steps of:

-   -   conjugating, or otherwise linking, a N-acylethanolamide compound        (e.g., a parent N-acylethanolamide compound) to a linker moiety;    -   conjugating, or otherwise linking, a moiety to the        linker-N-acylethanolamide moiety.

In some embodiments, the present disclosure provides methods ofmanufacturing a provided N-acylethanolamide derivative compound, whichmethod comprising steps of:

-   -   conjugating, or otherwise linking, a moiety to a linker moiety:    -   conjugating, or otherwise linking, a N-acylethanolamide compound        (e.g., a parent N-acylethanolamide compound) to the linker        moiety.

In some embodiments, a moiety is selected from the group consisting ofphosphate, butyric acid, glycerol, succinate, caprylic acid, gluconoicacid, eicosapentaeonoic acid, linoleic acid, succinate, and sucrosemoieties, and combinations thereof.

In some embodiments, the present disclosure provides method ofmanufacturing a provided pharmaceutical composition, which methodcomprising a step of:

-   -   formulating a provided N-acylethanolamide derivative compound        together with at least one pharmaceutically acceptable carrier.

EXEMPLIFICATION Example 1: (N-Palmiloylethanolamide) PEA in Pain

This Example provides a meta-analysis of 12 PEA pain studies and shows,among other things, that 67% of treated patients versus 21% of placeboachieved a VAS scores <=3. See FIGS. 2A-2F, and the table below, quotedfrom Guida et al., Dolor 2010; Paladini et al., Pain Physician Jounrla,February 2016; and Cobellis et al., Eur. J. Ob. and Gyn, July 2010.

Etiopathogenesis Degenerative Neuropathic Mixed Miscellaneous Patient1174 (79.1%) 170 (11.5%) 82 (5.5%) 58 (3.9%) number

Example 2: Treating IBS-D

The present Example illustrates treatment of IBS-D according to someembodiments of the present disclosure.

The present disclosure provides a strong rationale to combine butyricacid (BA) and PEA into a single dual active prodrug that will bemetabolized in the gut to the two active constituents that willalleviate pain and improve fecal consistency

In some embodiments, IBS-D treatment success comprises simultaneousimprovement in (i) daily worst abdominal pain score by >30% as comparedto baseline weekly average; and (ii) reduction in the Bristol StoolScale (BSS) to <5 on at least 50% of the days within a 12-week timeinterval. Alternatively or additionally, treatment success may be orcomprise improvement in daily worst abdominal pain in the absence of aconcurrent bowel movement. See FIGS. 2G-2Q, and the tables below, quotedfrom Scarpellini et al., Digestive Liver Disease, 2007; Banasiewicz etal., Colorectal Disease. 2012; Scheppach et al., Gastroenterology, 1992;Capasso et al., Br. J. of Pharm, 2014; and Borrelli et al., Br. J. ofPharm, 2015. For example, short-chain fatly acid irrigation has beenshown to ameliorate inflammation in diversion colitis. In the study byScheppach, et al., the effect of butyrate enemas was tested in 10patients with distal ulcerative colitis who had been unresponsive to orintolerant of standard therapy for 8 weeks. They were treated for 2weeks with sodium butyrate (100 mmol/L) and 2 weeks with placebo inrandom order (single-blind trial). Before and after treatment, clinicalsymptoms were noted and the degree of inflammation was gradedendoscopically and histologically. Rectal proliferation was assessed byautoradiography. After butyrate irrigation, stool frequency (n/day)decreased from 4.7±0.5 to 2.1±0.4 (P<0.01) and discharge of blood ceasedin 9 of 10 patients. The endoscopic score fell from 6.5±0.4 to 3.8±0.8(P<0.01). The histological degree of inflammation decreased from 2.4±0.3to 1.5±0.3 (P<0.02). Overall crypt proliferation was unchanged, but theupper crypt-labeling index fell from 0.086±0.019 to 0.032±0.003(P<0.03). On placebo, all of these parameters were unchanged. These datasupport the view that butyrate deficiency may play a role in thepathogenesis of distal ulcerative colitis and that butyrate irrigationameliorates this condition. Scheppach, et al. “Effect of Butyrate Enemason the Colonic Mucosa in Distal Ulcerative Colitis,” Gastroenterology,103:51-56 (1992).

Demographic Characteristics of Patients Included

IBS-CP IBS-CP (n = 28) (n = 22) P Gender (male/female) 8/20 8/14 ns Age[mean (SD)] 32 ± 5 34 ± 5 ns BMI (body mass index) 21 ± 8 22 ± 7 ns

Effects of Therapy on Severity of Symptoms

IBS-DP IBS-CP at after at after Symptom inclusion treatment inclusiontreatment Abdominal pain  9.5 (1)^(a) 6.1 (1)* 9.2 (1) 9.4 (1) Meteorism9.6 (1) 4.7 (1)* 9.3 (1) 9.0 (1) Flatulence 5.5 (1) 4.0 (1)* 5.1 (1) 5.1(1) ^(a)figure in brackets = SD *Statistically significant (p < 0.005).

MSB (N = 34) Placebo (N = 32) Number Percentage Number Percentage of ofstudy of of study patients group patients group P After 4 weeks of 11 322 6.25 <0.01 study Patients reporting subjective relief in IBS symptomsYES After 12 weeks of 18 53 5 15.6 <0.01 study Patients reportingsubjective relief in IBS symptoms YES

MSB (N = 34) Placebo (N = 32) Mean SD Median Mean SD Median P BaselineSpontaneous abdominal pain 0.53 0.51 1 0.53 0.51 1 ns Postprandialabdominal pain 0.44 0.50 0 0.44 0.50 0 ns Abdominal pain duringdefaecation 0.35 0.49 0 0.56 0.50 1 ns Urge sensation after thedefaection 0.26 0.45 0 0.38 0.49 0 ns Mucus in stool 0.15 0.36 0 0.130.34 0 ns Changes in stool consistency 0.44 0.50 0 0.38 0.49 0 nsConstipation 0.38 0.49 0 0.47 0.51 0 ns After 4 weeks of studySpontaneous abdominal pain 0.382 0.493 0 0.50 0.51 0.5 ns Postprandialabdominal pain 0.324 0.475 0 0.56 0.50 1 0.0968 Abdominal pain duringdefaecation 0.176 0.387 0 0.59 0.50 1 0.0032 Urge sensation afterdefaecation 0.235 0.431 0 0.41 0.50 0 ns Mucus in stool 0.088 0.288 00.13 0.34 0 ns Changes in stool consistency 0.382 0.493 0 0.41 0.50 0 nsConstipation 0.353 0.485 0.47 0.51 0 ns After 12 weeks of studySpontaneous abdominal pain 0.21 0.41 0 0.50 0.51 0.5 0.0132 Postprandialabdominal pain 0.21 0.41 0 0.56 0.50 1 0.0031 Abdominal pain duringdefaecation 0.15 0.36 0 0.59 0.50 1 0.0002 Urge sensation afterdefaecation 0.15 0.36 0 0.44 0.50 0 0.0100 Mucus in stool 0.12 0.33 00.22 0.42 0 ns Changes in stool consistency 0.18 0.39 0 0.41 0.50 00.0417 Constipation 0.24 0.43 0 0.47 0.51 0 0.0493 ns, not significant

There were no adverse effects in either arm o the study. Values areshown on proportions.

Example 3: Method Development, Plasma Stability, and MethodQualification for PEA and Prodrugs

The present example describes an LC-MS/MS method to determine PEA levelsin Sprague-Dawley rat plasma and to determine the stability ofcompounds(s) provided here.

An LC-MS/MS method for the determination of PEA and PEA-prodrug I-9 inSprague-Dawley rat plasma was developed. Each test article was infusedonto an ABSciex API4000 mass spectrometer to determine optimizedparameters. Next, liquid chromatography conditions were developed toobtain suitable specificity and to resolve the PEA and PEA-prodrugpeaks.

The stability of the PEA-prodrug I-9 was assessed in Sprague-Dawley ratplasma containing sodium heparin as the anticoagulant, and in acidifiedrat plasma containing citric acid and formic acid. The acidified plasmawas prepared by collecting Sprague-Dawley rat plasma over sodium heparinand adding 100 μL of 0.5 M citric acid per mL of blood. The blood wascentrifuged to generate plasma, and then 100 μL of 10% formic acid wasadded per mL of plasma.

PEA-prodrug was added to each matrix to a final concentration of 1μg/mL. Triplicate aliquots (50 μL) were immediately collected and addedto 150 μL of acetonitrile containing internal standard. The remainingplasma aliquots were split equally. One aliquot was allowed to stand atroom temperature while the second was placed on ice. After ninetyminutes, triplicate aliquots of each sample were collected and added toacetonitrile. The samples were centrifuged at 13000 rpm for ten minutes,and the resulting supernatant was analyzed using the developed LC-MS/MSmethod. The peak area response ratios (PARR) of the analyte and internalstandard from the incubated samples were then compared to the initialsample to determine the percent PEA prodrug remaining.

Following their initial analysis, the supernatant samples from theacidified plasma experiment were reinjected after storage on theautosampler (˜8° C.) for two hours in order to assess the stability ofthe PEA-prodrug in the post-extract matrix.

Results of the stability experiments are shown in Tables 3a, 3b, and 3c.

TABLE 3a Plasma Stability for PEA-Prodrug I-9 in Sprague Dawley RatPlasma Peak Area Average Peak Area % Response Ratio Response RatioRemaining Intial (t = 0) 2.78E−01 2.78E−01 ND 2.75E−01 2.81E−01 90minutes, ice 2.02E−01 2.04E−01 73.4% 2.03E−01 2.07E−01 90 minutes, RT8.38E−04 5.65E−04 0.203% 4.97E−04 3.60E−04 ND: not determined

TABLE 3b Plasma Stability for PEA-Prodrug I-9 in Acidified Sprague-DaleyRat Plasma Peak Area Average Peak Area % Response Ratio Response RatioRemaining Intial (t = 0) 1.31E+00 1.34E+00 ND 1.40E+00 1.31E+00 90minutes, ice 1.39E+00 1.43E+00  107% 1.46E+00 1.44E+00 90 minutes, RT1.23E+00 1.29E+00 96.0% 1.30E+00 1.33E+00 ND: not determined

TABLE 3c Post-Extract Stability for PEA-Prodrug I-9 Extracted fromAcidified Sprague-Dawley Rat Plasma Peak Area Average Peak Area %Response Ratio Response Ratio Remaining Intial (t = 0) 1.31E+00 1.34E+00ND 1.40E+00 1.31E+00 2 Hour Storage on 1.38E+00 1.42E+00 106%Autosampler (~8° C.) 1.46E+00 1.42E+00 ND: not determined

Results of the stability of in each matrix are presented in Tables 3aand 3b. The prodrug was found to be unstable when stored at roomtemperature (0.203% remaining) and on ice (73.4% remaining) for 90minutes. When the PEA-prodrug I-9 was fortified into rat plasmaacidified with citric and formic acids, it was found to be stable atroom temperature (107% remaining) and on ice (96.0% remaining) for 90minutes. In addition, it was demonstrated that the PEA-prodrug I-9 wasstable in the post-extract matrix following storage on the autosampler(˜8° C.) for two hours (Table 3c).

The specificity, accuracy, and precision of the method for PEA and thePEA-prodrug I-9 in acidified Sprague-Dawley rat plasma were evaluatedvia a single-day pre-study qualification. A single eight-point standardcurve and quality control samples at three levels with six replicateseach were extracted and analyzed for PEA or PEA-prodrug I-9. Inaddition, a dilution QC of a high concentration sample was prepared todemonstrate parallelism of the method. Standards and quality controlsamples were prepared from independently prepared stock solutions ofeach test compound. Results of the plasma qualification are presented inTables 3d and 3e.

TABLE 3d Method Qualification Results for PEA in Acidified Rat PlasmaNominal Conc. (ng/mL) 12.5 100 500 10000 Measured 10.3 105 509 10800Concentration 12.6 107 477 11800 (ng/mL) 11.6 110 451 10100 12.2 96.8496 13100 11.0 101 441 10900 14.0 109 448 10800 Average (ng/mL) 12.0 105470 11250 Accuracy (%) 95.6 105 94.1 113 CV (%) 10.9 4.83 5.96 9.39 n 66 6 6

TABLE 3e Method Qualification Results for PEA-Prodrug I-9 in AcidifiedRat Plasma Nominal Conc. (ng/mL) 2.50 50.0 500 10000 Measured 2.62 48.2537 10600 Concentration 2.12 50.2 517 10700 (ng/mL) 2.35 48.7 522 103002.39 49.5 536 10600 2.23 51.2 567 10900 2.15 48.2 547 10900 Average(ng/mL) 2.31 49.3 538 10667 Accuracy (%) 92.4 98.7 108 107 CV (%) 8.032.44 3.35 2.11 n 6 6 6 6

Plasma samples were extracted using the methods described below.

Analytical stock solutions (1.00 mg/mL of the free drug) were preparedin DMSO.

Sprague-Dawley rat blood was collected over sodium heparin and 0.5 Mcitric acid was added at a rate of 100 μL per mL of blood. Blood wascentrifuged to collect plasma. A 100 μL aliquot of 10% formic acid wasadded to each mL of plasma.

Standards were prepared in acidified rat plasma. Standards and qualitycontrol samples were prepared from independently prepared stocksolutions of each analyte. Working solutions were prepared in 50:50acetonitrile: water and then added to plasma to make calibrationstandards to final concentrations of 1000, 500, 100, 50, 10, 5, 1, and0.5 ng/mL and quality control samples to final concentrations of 2.50,50.0, and 500 ng/mL for PEA-prodrug I-9. For PEA, calibration standardswere prepared to final concentrations of 1000, 500, 100, 50, 25, 10, 5,and 2.5 ng/mL and quality control samples to final concentrations of12.50, 100, and 500 ng/mL. A high concentration dilution QC was preparedat 10,000 ng/mL for each analyte. This sample was diluted 20-fold intothe range of the assay prior to extraction.

Plasma samples were extracted via acetonitrile precipitation. Standardsand QCs: Add 10 μL of appropnate working solution to 50 μL of blankmatrix in a 96-well plate. Blanks: Add 10 μL 50:50 acetonitrile: waterto 50 μL of blank matrix in a 96-well plate. Samples: Add 10 μL 50:50acetonitrile: water to 50 μL of study sample in a 96-well plate. Cap andmix. Add 150 μL of acetonitrile (containing 100 ng/mL ritonavir as aninternal standard) to each well. Cap and mix at 1000 rpm for fiveminutes. Centrifuge the plate at 3000 rpm for ten minutes. Transfer a150 μL aliquot of the resulting supernatant into a clean 96-well plate.Cap for analysis. HPLC and Mass spectrometry conditions are described inTable 3f. Chromatograms are exemplified in FIGS. 3A, 3B, 3C, 3D, 3E, 3F,3G, and 3H.

TABLE 3f HPLC and Mass Spectrometry Conditions HPLC ConditionsInstrument: Waters ACQUITY UPLC Column: Waters HSS C₁₈, 30 × 2.1 mm id,1.8 μm Mobile Phase Buffer: 40 mM ammonium formate, pH 3.5 AqueousReservoir (A): 10% buffer, 90% water Organic Reservoir (B): 10% buffer,90% acetonitrile Gradient Program

TABLE 3-1 HPLC Gradient Program Time (mm) Grad. Curve % A % B 0.0 6 2080 0.75 6 0 100 0.80 6 50 50 1.0 6 50 50 Flow Rate: 800 μL/min InjectionVolume: 5 μL Run Time: 1.0 min Column Temperature: 40° C. SampleTemperature: 8° C. Strong Autosampler Wash: 1:1:1 (v:v:v)water:methanol:isopropanol with 0.2% formic acid Weak Autosampler Wash:4 mM ammonium formate Mass Spectrometer Conditions Instrument: PE SciexAPI4000 Interface: Electrospray (“Turbo Ion Spray” Mode: Multiplereactions monitoring (MRM) Gases: Cur 30, CAD 10, GS1 50, GS2 50 SourceTemperature: 500° C. Voltages and Ions Monitored*

TABLE 3-2 Mass Spectrometer Voltages and Ions Monitored PrecursorProduct Analyte Polarity Ion Ion IS DP EP CE CXP PEA Positive 300.2 62.15500 106 10 30 6 PEA- Positive 474.5 282.4 5500 80 10 24 7 prodrugRitonavir Positive 721.3 296.1 5500 65 10 25 18 (Internal STD) IS: IonSpray Voltage; DP: Declustering Potential; EP: Entrance Potential; CE:Collision Energy; CXP: Collision Cell Exit Potential; *All settings arein volts

Example 4: PEA Stability in Human and Rat Liver Microsomes, Human andRat Intestinal S9 Fractions, and Simulated Gastric Fluid

The present Example describes PEA stability observed in Human and RatLiver Microsomes, Human and Rat Intestinal S9 Fractions, and SimulatedGastnc fluid.

Liver Microsomal Stability

Mixed-gender human liver microsomes (Lot #1210347) and maleSprague-Dawley rat liver microsomes (Lot #1310030) were provided. Thereaction mixture, minus cofactors, was prepared as described below. Thetest article was added into the reaction mixture at a finalconcentration of 1 μM. The control compound, testosterone, was runsimultaneously with the test article in a separate reaction. An aliquotof the reaction mixture (without cofactor) was equilibrated in a shakingwater bath at 37° C. for 3 minutes. The reaction was initiated by theaddition of cofactor, and the mixture was incubated in a shaking waterbath at 37° C. Aliquots (100 μL) were withdrawn at 0, 10, 20, 30, and 60minutes. All samples were immediately combined with 400 μL of ice-cold50/50 acetonitrile/H₂O containing 0.1% formic acid and internal standardto terminate the reaction. The samples were then mixed and centrifugedto precipitate proteins. Calibration standards were prepared in matchedmatrix. All samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both the dosed prodrug I-9 and the expecteddrug (PEA). Analytical conditions are outlined in Appendix 4-1. The testarticle concentration at each time point was compared to the testarticle concentration at time 0 to determine the percent remaining ateach time point. Half-lives were calculated using GraphPad software,fitting to a single-phase exponential decay equation. Results are shownin Tables 4a and 4b.

Reaction Composition

Liver Microsomes 0.5 mg/mLNADPH (cofactor) 1 mMUDPGA (cofactor) 1 mM

Potassium Phosphate, pH 7.4 100 mM Magnesium Chloride 5 mM Test Article1 μM

TABLE 4a PEA stability observed in Human and Rat Liver MicrosomesCL_(int) ^(b) % Remaining of Initial (n = 1) Half- (mL/min/ Test 10 2030 60 Life^(a) mg Article Species 0 min min min min min (min) protein)I-9 Human 100 1.2 <1.0 <1.0 <1.0 <10(1.6) >0.139 (0.888) Rat 100 4.0<1.0 <1.0 <1.0 <10(2.2) >0.139 (0.642) ^(a)When the calculated half-lifeis longer than the duration of the experiment, the half-life isexpressed as > the longest incubation time. Similarly, if the calculatedhalf-life is less than the shortest time point, the half-life isexpressed as < that time point and the calculated half-life is alsolisted in parentheses. ^(b)Intrinsic clearance (CL_(int)) was calculatedbased on CL_(int) = k/P, where k is the elimination rate constant and Pis the protein concentration in the incubation.

Half- Cl_(int) Acceptable Control life (ml/min/mg Range Compound Species(min) protein) (t_(1/2), min) Testosterone Human 17 0.0792 ≤41 Rat 1.41.03 ≤15

TABLE 4b Measured concentrations of Prodrug and Drug Dosed Concentration(μM) Test 10 20 30 60 Article Species Analyte 0 min min min min min I-9Human I-9 0.18 0.0021 0 0 0 PEA 0.83 0.60 0.51 0.41 0.20 Rat I-9 0.210.0085 0 0 0 PEA 0.35 0.16 0.076 0.027 0.0030

Intestinal S9 Fraction Stability

Mixed-gender human intestinal S9 fraction (Lot #0710351) and maleSprague-Dawley rat intestinal S9 fraction (Lot #0510116) were provided.The reaction mixture, minus cofactors, was prepared as described below.The test article was added into the reaction mixture at a finalconcentration of 1 μM. The control compounds, testosterone and7-hydroxycoumarin, were run simultaneously with the test article in aseparate reaction. An aliquot of the reaction mixture (without cofactorcocktail) was equilibrated in a shaking water bath at 37° C. for 3minutes. The reaction was initiated by the addition of cofactorcocktail, and the mixture was incubated in a shaking water bath at 37°C. Aliquots (100 μL) were withdrawn at 0, 10, 20, 30, and 60 minutes.All samples were immediately combined with 400 uL of icecold 50/50acetonitrile/H₂O containing 0.1% formic acid and internal standard toterminate the reaction. The samples were then mixed and centrifuged toprecipitate proteins. Calibration standards were prepared in matchedmatrix. All samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both the dosed prodrug I-9 and the expecteddrug (PEA). Analytical conditions are outlined in Appendix 4-1. The testarticle concentration at each time point was compared to the testarticle concentration at time 0 to determine the percent remaining ateach time point. Half-lives were calculated using GraphPad software,fitting to single-phase exponential decay equation. Results are shown inTables 4c and 4d.

Reaction Composition

Intestinal S9 Fraction 1.0 mg/mLNADPH (cofactor) 1 mMUDPGA (cofactor) 1 mMPAPS (cofactor) 1 mMGSH (cofactor) 1 mM

Potassium Phosphate, pH 7.4 100 mM Magnesium Chloride 5 mM Test Article1 μM

TABLE 4c PEA stability observed in Human and Rat Intestinal S9 FractionCL_(int) ^(b) % Remaining of Initial (n = 1) Half- (mL/min/ Test 10 2030 60 Life^(a) mg Article Species 0 min min min min min (min) protein)I-9 Human 100 59 16 47 <1.0 <10(9.2) >0.0693 (0.0753) Rat 100 119 88 7549 55 0.125 ^(a)When the calculated half-life is longer than theduration of the experiment, the half-life is expressed as > the longestincubation time. Similarly, if the calculated half-life is less than theshortest time point, the half-life is expressed as < that time point andthe calculated half-life is also listed in parentheses. ^(b)Intrinsicclearance (CLint) was calculated based on CL_(int) = k/P, where k is theelimination rate constant and P is the protein concentration in theincubation.

Cl_(int) Control Half-life (ml/min/mg Compound Species (mm) protein)Testosterone Human 26 0.0269 Rat 116 0.00597 7-HC Human 7.3 0.943 Rat 340.0201

TABLE 4d Measured Concentrations of Prodrug and Drug Dosed Concentration(μM) Test 10 20 30 60 Article Species Analyte 0 min min min min min I-9Human I-9 0.61 0.36 0.099 0.029 0.0043 PEA 0.21 0.50 0.69 0.65 0.66 RatI-9 0.77 0.91 0.68 0.58 0.38 PEA 0.031 0.088 0.13 0.16 0.24

Simulated Gastric Fluid Stability

Studies were carried out in simulated gastric fluid (SGF). SGF wasprepared by dissolving 2.0 g of NaCl and 3.2 g of purified pepsin(derived from porcine stomach mucosa) in 7 mL of 10 N HCl and sufficientwater to make 1000 mL. The pH was adjusted to pH 1.2. Controlexperiments were also run without the addition of pepsin to the matrix.The test article was added into the SGF at 37° C. at a finalconcentration of 2 and incubated in a shaking water bath at 37° C.Individual tubes were dosed for each time point (0, 15, 30, 60, and 120minutes). At the appropriate time, 500 μL of ice-cold acetonitrilecontaining 0.1% formic acid and internal standard was added to a singletube. The starting time of each tube was staggered so that alltimepoints finished simultaneously. The samples were then mixed andcentrifuged. Calibration standards were prepared in matched matrix. Allsamples and standards were assayed by LCMS/MS using electrosprayionization for both the dosed prodrug I-9 and the expected drug (PEA).Analytical conditions are outlined in Appendix 4-1. The test articleconcentration at each time point was compared to the test articleconcentration at time 0 to determine the percent remaining at each timepoint. Half-lives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 4eand 4f.

TABLE 4e PEA stability observed in Simulated Gastric Fluid % Remainingof Initial (n = 1) Half- Test 15 30 60 120 Life^(a) Article Species 0min min min min min (min) I-9 SGF w/pepsin 100 95 80 92 86 >120 SGF w/opepsin 100 93 84 72 71 >120 ^(a)When the calculated half-life is longerthan the duration of the experiment, the half-life is expressed as > thelongest incubation time.

TABLE 4f Measured Concentrations of Prodrug and Drug Dosed Concentration(μM) Test 10 20 30 60 Article Matrix Analyte 0 min min min min min I-9Human I-9 1.52 1.45 1.21 1.40 1.31 PEA 0.0046 0.0078 0.0083 0.011 0.017Rat I-9 2.07 1.93 1.74 1.49 1.48 PEA 0.0046 0.0066 0.0067 0.0079 0.017

Microsome/S9 Fraction Control

As a control for the liver microsome and intestinal S9 fraction studies,additional experiments were run in reaction mixture, with the exclusionof microsomal proteins. The reaction mixture was prepared as describedbelow. The test article was added into the reaction mixture at a finalconcentration of 2 μM, and then incubated in a shaking water bath at 37°C. Individual tubes were dosed for each time point (0, 10, 20, 30, and60 minutes). At the appropriate time, 500 μL of ice-cold acetonitrilecontaining 0.1% formic acid and internal standard was added to a singletube. The starting time of each tube was staggered so that alltimepoints finished simultaneously. The samples were then mixed andcentrifuged to precipitate proteins. Calibration standards were preparedin matched matrix. All samples and standards were assayed by LC-MS/MSusing electrospray ionization for both the dosed prodrug I-9 and theexpected drug (PEA). Analytical conditions are outlined in Appendix 4-1.The test article concentration at each timepoint was compared to thetest article concentration at time 0 to determine the percent remainingat each time point. Half-lives were calculated using GraphPad software,fitting to a single-phase exponential decay equation. Results are shownin Table 4g and 4h.

Reaction Composition

NADPH (cofactor) 1 mMUDPGA (cofactor) 1 mMPAPS (cofactor) 1 mMGSH (cofactor) 1 mM

Potassium Phosphate, pH 7.4 100 mM Magnesium Chloride 5 mM Test Article2 μM

TABLE 4g Microsome/S9 Fraction Control % Remaining of Initial (n = 1)Half- Test 10 20 30 60 Life^(a) Article Matrix 0 min min min min min(min) I-9 Control 100 66 77 67 62 >60 ^(a)When the calculated half-lifeis longer than the duration of the experiment, the half-life isexpressed as > the longest incubation time.

TABLE 4h Measured Concentration of Prodrug and Drug Dosed Concentration(μM) Test 10 20 30 60 Article Species Analyte 0 min min min min min I-9Control I-9 2.24 1.47 1.72 1.5 1.38 PEA 0 0 0 0 0

Appendix 4-1 Liquid Chromatography

-   Column: Waters ACQUITY UPLC BEH C18 30×2.1 mm 1.7 μm-   M.P. Buffer: 25 mM ammonium formate buffer, pH 3.5-   Aqueous Reservoir (A): 90% water, 10% buffer-   Organic Reservoir (B): 90% acetonitrile, 10% buffer-   Flow Rate: 0.8 mL/minute

Gradient Program

TABLE 4-1 Liquid Chromatography Gradient Program Time (mm) % A % B 0.0020 80 0.75 0 100 0.80 50 50 1.00 50 50 Total Run Time: 1.0 minutesAutosampler: 10 μL Injection Volume Wash 1: water/methanol/2-propanol:1/1/1; with 0.2% formic acid Wash 2: 0.1% formic acid in water MassSpectrometer Instrument: PE SCIEX API 4000 Interface: Turbo IonsprayMode: Multiple reaction monitoring Method: 1.0 minute duration Settings:

TABLE 4-2 Mass Spectrometer Settings Test Article Q1/Q3 DP EP CE CXP ISTEM CAD CUR GS1 GS2 I-9 +474.5/282.4 80 10 24 7 5500 500 7 30 50 50 PEA+300.2/62.1  106 10 30 6 5500 500 7 30 50 50 Warfarin(IS) +309.2/251.180 10 30 5 5500 500 7 30 50 50

Example 5: Determination of the Oral Bioavailability of PEA FollowingAdministration of PEA and PEA-Prodrug in Male Sprague-Dawley Rats

The present Example describes PEA levels observed in Sprague-Dawley ratssamples after oral administration of compound(s) provided herein.

The oral bioavailability of palmitoylethanolamide (PEA) was evaluatedfollowing oral dosing of PEA, a marketed PEA product, Normast, or aPEA-prodrug I-9. PEA was also dosed intravenously at 1 mg/kg. Bloodsamples were collected up to 8 hours post-dose, and PEA and PEA-prodrugplasma concentrations were determined with a qualified LC-MS/MS method.Pharmacokinetic analysis was conducted by a non-compartmental modelusing Phoenix WinNonlin v.6.4 software.

Preparation of Dosing Formulations

PEA for IV and PO dosing (St. Louis, Mo.). PEA-prodrug (lot 261-SB-85)and Normast (Epitech Group, lot D106C6) were provided. The IV dosingsolution was prepared fresh on the day of dosing at 0.5 mg/mL in avehicle comprised of 10% solutol HS15, 10% n-methylpyrrolidone (NMP),10% polyethylene glycol 400 (PEG400), and 70% water. For PO dosing,torpac capsules were loaded with an appropriate amount of PEA,PEA-prodrug, or normast powder. Doses in groups 2, 3, and 4 wereprepared to deliver a similar amount of active drug per rat. ThePEA-prodrug is 63.2% (w/w) active and Normast contains 72.7% active(w/w). The prodrug dose in group 5 was the maximum amount of powder thatwould fit into a single capsule.

The pharmacokinetics of PEA and the PEA-prodrug were evaluated in fastedmale Sprague-Dawley rats. Rats were housed one per cage. Each rat wasfitted with a jugular vein cannula (JVC) for blood collection. Ratsintended for IV dosing were fitted with an additional JVC for dosing.Each study group was dosing in triplicate. Rats were fasted for aminimum of twelve hours prior to dosing. Food was returned at four hourspost dosing. Animals had free access to water throughout the study.

Animal Dosing

Blood samples (˜300 μL) were collected from the rats via a JVC andplaced into chilled polypropylene tubes containing sodium heparin as ananticoagulant, and 30 μL of 0.5 M citric acid. Samples were maintainedchilled throughout processing. Blood samples were centrifuged at 4° C.and 3,000 g for 5 minutes. Plasma (˜150 μL) was then transferred to achilled, labeled polypropylene tube containing 15 μL of 10% formic acid,placed on dry ice, and stored in a freezer maintained at −60° C. to −80°C. Blood sampling times are shown in Table 5a.

TABLE 5a Study Design Dosing Blood Formulation Dosing Sampling Dose TestNo. of Dosing Dose Conc. Volume Time Group Article Animals Route (mg/kg)(mg/mL) (mL/kg) Vehicle Points 1 PEA 3 IV   1 0.5 2 10% Pre-dose,Solutol 5, 15, HS15, 10% 30 min, NMP, 10% 1, 2, PEG400, 4 and 70% water8 hours 2 PEA 3 PO ~10  NA 1 capsule Torpac Pre-dose, capsule 15, 3 PEA-3 PO ~16* NA 1 capsule Torpac 30 min, prodrug capsule 1, 2, 4 RLD 3 PO~16* NA 1 capsule Torpac 4, and (Normast) capsule 8 hours 5 PEA- 3 PO~126*  NA 1 capsule Torpac prodrug capsule NMP: n-methyl pyrrolidone;*mg of actual pro-drug or drug product, not corrected for activecontent.

An LC-MS/MS method for the determination of PEA and PEA-prodrug isdescribed above (see e.g., Example 3).

Pharmacokinetic parameters were calculated from the time course of theplasma concentration and are presented in Tables 3 through 9.Pharmacokinetic parameters were determined with Phoenix WinNonlin (v6.4)software using a non-compartmental model. Maximum plasma concentrations(C₀) after IV dosing were estimated by extrapolation of the first twotime points back to t=0. Maximum plasma concentration (C_(max)) and thetime to reach maximum plasma drug concentration (T_(max)) after oraldosing were observed from the data. Area under the time concentrationcurve (AUC) was calculated using the linear trapezoidal rule withcalculation to the last quantifiable data point, and with extrapolationto infinity if applicable. At least three quantifiable data points wererequired to determine the AUC. Plasma half-life (t_(1/2)) was calculatedfrom 0.693/slope of the terminal elimination phase. Mean residence time,MRT, was calculated by dividing the area under the moment curve (AUMC)by the AUC. Clearance (CL) was calculated from dose/AUC. Steady-statevolume of distribution (V_(ss)) was calculated from CL*MRT (meanresidence time). Bioavailability was determined by dividing theindividual PO dose normalized AUC_(last) values by the average IVAUC_(last) value. Any samples below the limit of quantitation weretreated as zero for pharmacokinetic data analysis.

The IV dosing solution was analyzed by LC-MS/MS. The measured dosingsolution concentration is shown in Table 5b. The dosing solutions werediluted into rat plasma and analyzed in triplicate. All concentrationsare expressed as mg/mL of the free base. Capsules were not analyzed.Nominal dosing concentrations were used in all calculations for thesegroups.

TABLE 5b Measured Dosing Solution Concentrations (mg/mL). MeasuredNominal Dosing Route of Dosing Solution Test Adminis- Conc. Conc. % ofArticle tration Vehicle (mg/mL) (mg/mL) Nominal PEA IV 10% Solutol 0.50.382 76.3 HS15, 10% NMP, 10% PEG400, 70% water NMP: n-methylpyrrolidone

Endogenous levels of PEA were found in all rats. Measured concentrationsof PEA in plasma samples were corrected by subtracting the concentrationof PEA measured in the pre-dose samples. These corrected values arereported in the tables below and were used to determine pharmacokineticparameters. Any corrected values that were negative are reported as notdetermined (ND).

Following IV dosing at 1 mg/kg, PEA had an average half-life of0.596±0.165 hours, an average clearance rate of 15.1±3.15 L/hr/kg and anaverage volume of distribution of 9.12±0.832 L/kg. Results are shown inTable 5c and FIGS. 4A and 4B.

TABLE 5c Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Intravenous Administration ofPEA in Male Sprague-Dawley Rats at 1 mg/kg (Group 1). Intravenous (1mg/kg) Rat # Time (hr) 907 908 909 Mean SD 0 (pre-dose) 10.3 6.96 6.507.92 2.07 0.083 122 137 174 144 26.6 0.25 47.2 49.6 47.7 48.2 1.29 0.5027.5 30.7 35.5 31.2 4.02 1.0 9.60 7.94 16.1 11.2 4.31 2.0 5.20 5.64 9.906.91 2.60 4.0 ND ND ND ND ND 8.0 ND ND ND ND ND Animal Weight (kg) 0.2750.265 0.262 0.267 0.007 Volume Dosed (mL) 0.55 0.53 0.52 0.53 0.02 C₀(ng/mL)¹ 196 227 331 251 70.9 t_(max) (hr)¹ 0 0 0 0 0 t_(1/2) (hr) 0.5640.449 0.774 0.596 0.165 MRT_(last) (hr) 0.399 0.374 0.435 0.402 0.0306CL (L/hr/kg) 17.4 16.4 11.5 15.1 3.15 V_(ss) (L/kg) 10.0 8.39 8.94 9.120.832 AUC_(last) (hr ng/mL) 53.3 57.2 75.8 62.1 012.0 AUC_(∞) (hr ng/mL)57.6 60.8 86.8 68.4 16.0 C₀: maximum plasma concentration extrapolatedto t = 0; t_(max): time of maximum plasma concentration; t_(1/2):half-life, data points used for half-life determination are in bold;MRT_(last): mean residence time, calculated to the last observable timepoint; CL: clearance; V_(ss): steady state volume of distribution;AUC_(last): area under the curve, calculated to the last observable timepoint; AUC_(∞): area under the curve, extrapolated to infinity; ND: notdetermined; ¹Extrapolated to t = 0.

Following oral dosing of PEA in group 2 (average dose of 11.7 mg/kg),nearly all plasma samples were below the endogenous levels measured inthe predose samples. The highest concentration measured in one animalwas 5.30 ng/mL at 2 hours post dose. No AUCs or bioavailability valuescould be determined for this group. Results are shown in Table 5d.

TABLE 5d Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA After Oral Administration of PEA inMale Sprague-Dawley Rats at ~10 mg/kg (Group 2). Oral (~10 mg/kg, PEAafter PEA Dose) Rat # Time (hr) 910 911 912 Mean SD 0 (pre-dose) 8.048.30 6.00 7.45 1.26 0.25 ND ND ND ND ND 0.50 ND ND ND ND ND 1.0 ND ND2.34 ND ND 2.0 ND ND 5.30 ND ND 4.0 ND ND ND ND ND 8.0 ND ND ND ND NDAnimal Weight (kg) 0.250 0.260  0.262 0.257 0.006 Volume Dosed (mg) 3.03.0 3.0  3.0 0.0 Dose (mg/kg) 12.0 11.5 11.5  11.7 0.295 C_(max) (ng/mL)ND ND 5.30 ND ND t_(max) (hr) N ND 2.0  ND ND t_(1/2) (hr) ND ND ND NDND MRT_(last) (hr) ND ND ND ND ND AUC_(last) (hr ng/mL) ND ND ND ND NDAUC_(∞) (hr ng/mL) ND ND ND ND ND Dose-normalized Values AUC_(last) (hrkg ng/mL/mg) ND ND ND ND ND AUC_(∞) (hr kg ng/mL/mg) ND ND ND ND NDBioavailability (%) ND ND ND ND ND C₀: maximum plasma concentration;t_(max): time of maximum plasma concentration; t_(1/2): half-life, datapoints used for half-life determination are in bold; MRT_(last): meanresidence time, calculated to the last observable time point;AUC_(last): area under the curve, calculated to the last observable timepoint; AUC_(∞): area under the curve, extrapolated to infinity; ND: notdetermined.

Following oral dosing of the PEA-prodrug in group 3 (average dose of11.7 mg/kg active equivalents), nearly all plasma samples were below theendogenous levels measured in the predose samples. The highestconcentration measured in one animal (rat 914) was 0.630 ng/ml at 0.25hours post dose. The bioavailability in this animal was 0.108%. Resultsare shown in Table 5e and 5f.

TABLE 5e Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA-Prodrug After Oral Administration ofPEA-Prodrug in Male Sprague-Dawley Rats at ~16 mg/kg (Group 3). Oral(~16 mg/kg, PEA-prodrug after PEA-prodrug Dose) Rat # Time (hr) 913 914915 Mean SD 0 (pre-dose) BLOQ BLOQ BLOQ ND ND 0.25 BLOQ BLOQ BLOQ ND ND0.50 BLOQ BLOQ BLOQ ND ND 1.0 BLOQ BLOQ BLOQ ND ND 2.0 BLOQ BLOQ BLOQ NDND 4.0 BLOQ BLOQ BLOQ ND ND 8.0 BLOQ BLOQ BLOQ ND ND Animal Weight (kg)0.251 0.266 0.259 0.259 0.008 Amount Dosed (mg) 4.8 4.8 4.8 4.8 0 Dose(mg/kg) 19.1 18.0 18.5 18.6 0.540 C_(max) (ng/mL) ND ND ND ND ND t_(max)(hr) ND ND ND ND ND t_(1/2) (hr) ND ND ND ND ND MRT_(last) (hr) ND ND NDND ND AUC_(last) (hr ng/mL) ND ND ND ND ND AUC_(∞) (hr ng/mL) ND ND NDND ND Dose-normalized Values AUC_(last) (hr kg ng/mL/mg) ND ND ND ND NDAUC_(∞) (hr kg ng/mL/mg) ND ND ND ND ND C_(max): maximum plasmaconcentration; t_(max): time of maximum plasma concentration; t_(1/2):half-life, data points used for half-life determination are in bold;MRT_(last): mean residence time, calculated to the last observable timepoint; AUC_(last): area under the curve, calculated to the lastobservable time point: AUC_(∞): area under the curve, extrapolated toinfinity; ND: not determined. BLOQ: below the limit of quantitation (0.5ng/mL).

TABLE 5f Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA After Oral Administration of PEAProdrug in Male Sprague-Dawley Rats at ~16 mg/kg (Group 3). Oral (~16mg/kg, PEA after PEA-prodrug Dose) Rat # Time (hr) 913 914 915 Mean SD 0(pre-dose) 6.23 3.80  5.17 5.07 1.22 0.25 ND 0.630 ND ND ND 0.50 ND ND ND ND ND 1.0 ND 0.500 ND ND ND 2.0 ND ND  ND ND ND 4.0 ND 0.230 ND ND ND8.0 ND ND  ND ND ND C_(max) (ng/mL) ND 0.630 ND ND ND t_(max) (hr) ND0.25  ND ND ND t_(1/2) (hr) ND ND³ ND ND ND MRT_(last) (hr) ND 1.75  NDND ND AUC_(last) (hr ng/mL) ND 0.763 ND ND ND AUC_(∞) (hr ng/mL) ND ND³ND ND ND Dose-normalized Values¹ AUC_(last) (hr kg ng/mL/mg) ND  0.0669ND ND ND AUC_(∞) (hr kg ng/mL/mg) ND ND³ ND ND ND Bioavailability (%)²ND 0.108 ND ND ND C_(max): maximum plasma concentration; t_(max): timeof maximum plasma concentration; t_(1/2): half-life, data points usedfor half-life determination are in bold; MRT_(last): mean residencetime, calculated to the last observable time point; AUC_(last): areaunder the curve, calculated to the last observable time point; AUC_(∞):area under the curve, extrapolated to infinity; ND: not determined;¹dose normalized values determined by dividing the parameter by the dosein mg/kg; ²bioavailability determined by dividing the individual dosenormalized AUC_(last) value by the average IV AUC_(last) value; ³notdetermined due to lack of quantifiable data points trailing the C_(max).

Following oral dosing of Normast in group 4 (average dose of 18.5mg/kg), many plasma samples were below the endogenous levels measured inthe predose samples. Average maximum plasma concentration (n=3) was3.38±2.17 ng/mL. Average bioavailability (n=2) was 0.561%. Results areshown in Table 5g.

TABLE 5g Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA After Oral Administration of Normastin Male Sprague-Dawley Rats at ~16 mg/kg (Group 4). Oral (~16 mg/kg, PEAafter Normast Dose) Rat # Time (hr) 916 917 918 Mean SD 0 (pre-dose)4.43 2.36 4.05 3.61 1.10 0.25 ND  0.200 ND  ND ND 0.50 ND ND  2.19 ND ND1.0 1.11 ND  5.83 3.47 ND 2.0 ND  0.960 2.10 1.53 ND 4.0 2.62 1.69 ND 2.16 ND 8.0 ND ND  ND  ND ND Animal Weight (kg)  0.264 0.25  0.263 0.259 0.008 Amount Dosed (mg) 4.8  4.8  4.8  4.8  0.0 Dose (mg/kg)18.2  19.2  18.3  18.5  0.569 C_(max) (ng/mL) 2.62 1.69 5.83 3.38 2.17t_(max) (hr) 4.0  4.0  1.0  3.0  1.7 t_(1/2) (hr) ND ND³ ND³ ND NDMRT_(last) (hr) ND 3.04 1.10 2.07 ND AUC_(last) (hr ng/mL) ND 3.18 6.244.71 ND AUC_(∞) (hr ng/mL) ND ND³ ND³ ND ND Dose-normalized Values¹AUC_(last) (hr kg ng/mL/mg) ND  0.227  0.469  0.348 ND AUC_(∞) (hr kgng/mL/mg) ND ND³ ND³ ND ND Bioavailability (%)² ND  0.366  0.756  0.561ND C_(max): maximum plasma concentration; t_(max): time of maximumplasma concentration; t_(1/2): half-life, data points used for half-lifedetermination are in bold; MRT_(last): mean residence time, calculatedto the last observable time point; AUC_(last): area under the curve,calculated to the last observable time point; AUC_(∞): area under thecurve, extrapolated to infinity; ND: not determined; ¹dose normalizedvalues determined by dividing the parameter by the dose in mg/kg;²bioavailability determined by dividing the individual dose normalizedAUC_(last) value by the average IV AUC_(last) value; ³not determined dueto lack of quantifiable data points trailing the C_(max).

Following oral dosing of the PEA-prodrug in group 5 (average dose of90.6 mg/kg active equivalents), many plasma samples were below theendogenous levels measured in the predose samples. Average maximumplasma concentration (n=3) was 2.52±0.829 ng/mL. Bioavailabilitydetermined in one animal was 0.124%. Results are shown in Table 5h and5j.

TABLE 5h Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA-Prodrug After Oral Administration ofPEA-Prodrug in Male Sprague-Dawley Rats at ~126 mg/kg (Group 5) Oral(~126 mg/kg, PEA-prodrug after PEA-prodrug Dose) Rat # Time (hr) 919 920921 Mean SD 0 (pre-dose) BLOQ BLOQ BLOQ ND ND 0.25 BLOQ BLOQ BLOQ ND ND0.50 BLOQ BLOQ BLOQ ND ND 1.0 BLOQ BLOQ BLOQ ND ND 2.0 BLOQ BLOQ BLOQ NDND 4.0 BLOQ BLOQ BLOQ ND ND 8.0 BLOQ BLOQ BLOQ ND ND Animal Weight (kg)0.263 0.269 0.263 0.265 0.003 Amount Dosed (mg) 38 38 38 38 0 Dose(mg/kg) 144 141 144 143 1.86 C_(max) (ng/mL) ND ND ND ND ND t_(max) (hr)ND ND ND ND ND t_(1/2) (hr) ND ND ND ND ND MRT_(last) (hr) ND ND ND NDND AUC_(last) (hr ng/mL) ND ND ND ND ND AUC_(∞) (hr ng/mL) ND ND ND NDND Dose-normalized Values AUC_(last) (hr kg ng/mL/mg) ND ND ND ND NDAUC_(∞) (hr kg ng/mL/mg) ND ND ND ND ND C_(max): maximum plasmaconcentration; t_(max): time of maximum plasma concentration; t_(1/2):half-life, data points used for half-life determination are in bold;MRT_(last): mean residence time, calculated to the last observable timepoint; AUC_(last): area under the curve, calculated to the lastobservable time point; AUC_(∞): area under the curve, extrapolated toinfinity; ND: not determined; BLOQ: below the limit of quantitation (0.5ng/mL).

TABLE 5j Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA After Oral Administration of PEAProdrug in Male Sprague-Dawley Rats at ~126 mg/kg (Group 5). Oral (~126mg/kg, PEA after PEA-prodrug Dose) Rat # Time (hr) 919 920 921 Mean SD 0(pre-dose) 1.82 1.57 2.40 1.93 0.426 0.25 ND ND ND ND ND 0.50 ND ND NDND ND 1.0 ND 3.47 ND ND ND 2.0  0.690 1.84  0.450  0.993 0.743 4.0 1.931.49 2.17 1.86 0.345 8.0 ND ND ND ND ND C_(max) (ng/mL) 1.93 3.47 2.172.52 0.829 t_(max) (hr) 4.0  1.0  4.0  3.0  1.7  t_(1/2) (hr) ND ND NDND ND MRT_(last) (hr) ND 2.06 ND ND ND AUC_(last) (hr ng/mL) ND 6.85 NDND ND AUC_(∞) (hr ng/mL) ND ND ND ND ND Dose- normalized Values¹AUC_(last) (hr kg ND  0.0767 ND ND ND ng/mL/mg) AUC_(∞) (hr kg ng/mL/mg)ND ND ND ND ND Bioavailability (%)² ND  0.124 ND ND ND C_(max): maximumplasma concentration; t_(max): time of maximum plasma concentration;t_(1/2): half-life, data points used for half-life determination are inbold; MRT_(last): mean residence time, calculated to the last observabletime point; AUC_(last): area under the curve, calculated to the lastobservable time point; AUC_(∞): area under the curve, extrapolated toinfinity; ND: not determined; ¹dose normalized values determined bydividing the parameter by the dose in mg/kg; ²bioavailability determinedby dividing the individual dose normalized AUC_(last) value by theaverage IV AUC_(last) value; ³not determined due to lack of quantifiabledata points trailing the C_(max).

The PEA-prodrug was not detectable in any plasma samples. Nopharmacokinetic parameters were determined for the PEA-prodrug.

Example 6: PEA Levels in Rabbit Plasma Samples

The present Example describes PEA levels observed in rabbit plasmasamples after oral administration of compound(s) provided herein. Bloodsamples were taken approximately five hours after the morning dosing.

Instability of the PEA-prodrug I-9 in untreated plasma samples has beendemonstrated previously (see, e.g., Examples 3-5). Plasma samples fromthe current study were not treated to prolong the post-collectionstability of the prodrug. Instead, all samples were allowed to stand,thawed and untreated, at room temperature for four hours prior toextraction and analysis. This strategy allowed for any remaining prodrugin the sample to convert to PEA. All reported concentrations are totalPEA in the sample.

Seventy-nine New Zealand White rabbit plasma samples were analyzed witha previously developed LC-MS/MS method for the detection ofpalmitoylethanolamide (PEA). A total of 79 plasma samples were receivedfor analysis. All samples were received frozen in good condition.Samples were stored at −80° C. until analysis.

An LC-MS/MS method for the determination of PEA in plasma was used toquantify samples in this study. See, e.g., Example 3. Study samples wereextracted using the methods described in Examples 3-5.

Results are shown in Tables 6a through 6d

TABLE 6a Individual and Average PEA Concentrations (ng/ML) in Group 1(Vehicle treated) Plasma Samples. Animal ID Day R3145 R2038 R2769 R3147R3154 Average SD −5 BLOQ BLOQ BLOQ BLOQ BLOQ ND ND −3 BLOQ BLOQ BLOQBLOQ BLOQ ND ND −3 BLOQ BLOQ 2.77 BLOQ BLOQ ND ND 7 BLOQ BLOQ BLOQ BLOQBLOQ ND ND BLOQ: below the limit of quantitation (2.5 ng/mL); ND: notdetermined.

TABLE 6b Individual and Average PEA Concentrations (ng/mL) in Group 2(Normast treated at 32 mg/kg/day) Plasma Samples Animal ID Day R2771R3152 R3143 R3138 R3140 Average SD −5 BLOQ BLOQ BLOQ BLOQ BLOQ ND ND −3BLOQ BLOQ BLOQ BLOQ BLOQ ND ND −3 BLOQ 2.97 4.52 BLOQ BLOQ 3.75 ND 7BLOQ BLOQ BLOQ 5.29 BLOQ ND ND BLOQ: below the limit of quantitation(2.5 ng/mL); ND: not determined.

TABLE 6c Individual and Average PEA Concentrations (ng/mL) in Group 3(PEA-prodrug treated at 32 mg/kg/day) Plasma Samples Animal ID Day R3156R2050 R2833 R2037 R2770 Average SD −5 BLOQ BLOQ BLOQ BLOQ BLOQ ND ND −3BLOQ BLOQ BLOQ BLOQ BLOQ ND ND 3 6.64 8.70 10.8 4.14 6.63 ND ND 7 5.626.09 4.47 BLOQ 5.57 ND ND BLOQ: below the limit of quantitation (2.5ng/mL); ND: not determined.

TABLE 6d Individual and Average PEA Concentrations (ng/mL) in Group 4(PEA-prodrug at 160 mg/kg/day) Plasma Samples Animal ID Day R3141 R3139R3153 R3157 R3151 Average SD −5 BLOQ BLOQ BLOQ BLOQ BLOQ ND ND −3 BLOQBLOQ BLOQ BLOQ BLOQ ND ND 3 27.6 20.0 27.6 24.7 17.0 23.4 4.73 7 7.108.12 9.93 14.5 14.3 10.8 3.45 BLOQ: below the limit of quantitation (2.5ng/mL); ND: not determined.

One sample from the vehicle group (Group 1) is reported with ameasureable PEA concentration (2.77 ng/mL, Day 3, Animal R2769). Thispositive result is likely due to the fact that PEA is an endogenousfatty acid. Several samples from the vehicle group and predose timepoints were near the LLOQ of the method (2.5 ng/mL), but this was theonly such sample that exceeded the LLOQ.

Example 7: Determination of Bioavailability of PEA Following Oral (PO)Administration of PEA-Prodrugs in Male Sprague-Dawley Rats

The present Example describes oral bioavailability of PEA followingadministration of PEA prodrugs I-2, I-3, I-5, I-6, I-7, I-9, and I-11 inmale Sprague-Dawley rats.

Oral bioavailability of palmitoylethanolamide (PEA) was evaluatedfollowing oral dosing of seven different PEA-prodrugs. Prodrugs weredosed orally to deliver a total PEA dose of 10 mg/kg. Following oraladministration of the PEA-prodrugs, PEA plasma concentrations weredetermined with a qualified LC-MS/MS method.

Preparation of Dosing Formulations

Pro-drugs were dosed so that a total dose of 10 mg/kg of PEA wasadministered. Each prodrug was formulated in a vehicle comprised of 10%Solutol, 10% n-methyl pyrrolidone (NMP), 10% polyethylene glycol 400(PEG400) and 70% water. Formulations were prepared fresh on the day ofdosing.

Animal Dosing

Pharmacokinetics of PEA were evaluated in fasted male Sprague-Dawleyrats. Rats were housed one per cage. Each rat was fitted with a jugularvein cannula (JVC) for blood collection. Each study group was dosing intriplicate. Rats were fasted for a minimum of twelve hours prior todosing. Food was returned at four hours post dosing. Animals had freeaccess to water throughout the study.

Blood samples (˜300 μL) were collected from the rats via a JVC andplaced into chilled polypropylene tubes containing sodium heparin as ananticoagulant, and 30 μL of 0.5 M citric acid. Samples were maintainedchilled throughout processing. Blood samples were centrifuged at 4° C.and 3,000 g for 5 minutes. Plasma (˜150 μL) was then transferred to achilled, labeled polypropylene tube containing 15 μL of 10% folinicacid, placed on dry ice, and stored in a freezer maintained at −60° C.to −80° C. Blood sampling times are shown in Table 7a.

TABLE 7a Study Design Dose Dosing Blood Number (mg/kg Solution DosingSampling Dose Test of Dosing of pro- Conc. Volume Time Group ArticleAnimals Route drug)* (mg/mL) (mL/kg) Vehicle Points 1 I-9 3 PO 16 3 5.310% Pre-dose, 2 I-6 3 PO 19 3 6.3 Solutol, 5, 15, 3 I-5 3 PO 19.7 3 6.610% NMP, 30 min, 4 I-3 3 PO 24.5 3 8.2 10% 1, 2, 5 I-2 3 PO 12.5 3 4.2PEG400, 4, and 6 I-11 3 PO 20.7 3 6.9 70% water 8 hours 7 I-7 3 PO 24.53 8.2 NMP: n-methyl pyrrolidone; *dose of actual pro-drug, all deliver10 mg/kg of PEA.

An LC-MS/MS method for determination of PEA and PEA-prodrug is describedabove (see e.g., Example 3).

Pharmacokinetic parameters were calculated from the time course of theplasma concentration and are presented in Tables 7b-7h and FIGS. 7A-7N.Maximum plasma concentration (C_(max)) and time to reach maximum plasmadrug concentration (T_(max)) after oral dosing were observed from thedata. Area under the time concentration curve (AUC) was calculated usingthe linear trapezoidal rule with calculation to the last quantifiabledata point, and with extrapolation to infinity if applicable. At leastthree quantifiable data points were required to determine AUC. Plasmahalf-life (t_(1/2)) was calculated from 0.693/slope of the terminalelimination phase. Mean residence time, MRT, was calculated by dividingarea under the moment curve (AUMC) by AUC. Bioavailability wasdetermined by dividing individual dose-normalized PO AUC_(last) valuesby the average IV AUC_(last) value (IV data Example 5). Samples belowthe limit of quantitation were treated as zero for pharmacokinetic dataanalysis.

Results

No adverse reactions were observed following the oral administration ofPEA pro-drugs in male Sprague-Dawley Rats in this study.

The dosing solutions were not analyzed by LC-MS/MS. Concentrations areexpressed as mg/ml of the free base. Nominal dosing level was used inall calculations.

Individual and average plasma concentrations and pharmacokineticparameters for PEA and are shown in Tables 7b-7h. Data are expressed asng/mL of the free drug. Samples that were below the limit ofquantitation were not used in the calculation of averages. Plasmaconcentration versus time data are plotted in FIGS. 7A-7N. Endogenouslevels of PEA were found in all rats. Measured concentrations of PEA inplasma samples were corrected by subtracting the concentration of PEAmeasured in the pre-dose samples. These corrected values are reported inthe tables below and were used to determine pharmacokinetic parameters.Any corrected values that were negative are reported as not determined(ND).

Following PO dosing of I-9 (Group 1), maximum plasma concentrations(average of 10.4±2.29 ng/mL) were observed at 1 hour post dosing.Average half-life was not determined due to a lack of quantifiable datapoints trailing the C_(max). Average exposure based on thedose-normalized AUC_(last) was 1.61±0.692 hr*kg*ng/′mL/mg. Based on theIV data from study Example 5, the average oral bioavailability for I-9was 2.60±1.11%. Results are shown in Table 7b and FIGS. 5A and 5B.

TABLE 7b Individual and Average Plasma Concentrations (ng/ml) andPharmacokinetic Parameters for PEA after Oral Administration of I-9 at16/mg/kg in Male Sprague-Dawley Rats. Oral (16 mg/kg I-9 equals 10 mg/kgPEA) Rat # Time (hr) 161 162 163 Mean SD 0 (pre-dose) 2.74 3.83 3.303.29  0.545 0.083 1.83 ND   0.170 1.00 ND 0.25 7.56 2.41 5.50 5.16 2.590.50 7.36 7.07 11.4  8.61 2.42 1.0 9.36 8.77 13.0  10.4  2.29 2.0 3.931.84 5.33 3.70 1.76 4.0 ND  ND   0.640 ND ND 8.0 ND  ND  ND  ND NDAnimal Weight (g)  0.259  0.258  0.260  0.259  0.001 Amount Dosed (m:)1.37 1.37 1.38 1.37 0.01 C_(max) (ng/mL) 9.36 8.77 13.0  10.4  2.29t_(max) (hr) 1.00 1.00 1.00 1.00 0.00 t_(1/2) (hr) ND³ ND³ ND³ ND NDMRT_(last) (hr)  0.933  0.913 1.28 1.04  0.208 AUC_(last) (hr ng/mL)13.7  10.8  24.0  16.1  6.92 AUC_(∞) (hr ng/mL) ND³ ND³ ND³ ND NDDose-normalized Values¹ AUC_(last) (hr kg 1.37 1.08 2.40 1.61  0.692ng/mL/mg) AUC_(∞) (hr kg ng/mL/mg) ND³ ND³ ND³ ND ND Bioavailability(%)² 2.20 1.74 3.86 2.60 1.11 C_(max): maximum plasma concentration;t_(max): time of maximum plasma concentration; t_(1/2): half-life, datapoints used for half-life determination are in bold; MRT_(last): meanresidence time, calculated to the last observable time point;AUC_(last): area under the curve, calculated to the last observable timepoint; AUC_(∞): area under the curve, extrapolated to infinity; ND: notdetermined; BLOQ: below the limit of quantitation (0.5 ng/mL); ¹Dosenormalized by dividing the parameter by the nominal dose in mg/kg;²Bioavailability determined by dividing the individual dose normalizedoral AUC_(last) values by the average IV AUC_(last) value 62.1 hr*ng/mLfrom Example 5; ³not determined due to lack of quantifiable data pointstrailing the C_(max).

Following PO dosing of I-6 (Group 2), maximum plasma concentrations(average of 11.7±5.39 ng/mL) were observed at fifteen minutes postdosing. Average halflife after PO dosing was 1.96±2.04 hours. Averageexposure based on the dose-normalized AUC_(last) was 1.40±0.737hr*kg*ng/mL/mg. Based on the IV data from Example 5, average oralbioavailability for I-6 was 2.25±1.19%. Results are shown in Tables 7cand FIGS. 5C and 5D.

TABLE 7c Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-6 at19 mg/kg in Male Sprague-Dawley Rats (Group 21. Oral (19 mg/kg I-6equals 10 mg/kg PEA) Rat # Time (hr) 164 165 166 Mean SD 0 (pre-dose)3.27 4.06 4.26 3.86  0.523 0.083 2.13 1.27 ND 1.70 ND 0.25 17.3 11.36.54 11.7 5.41 0.50 16.5 7.24 5.73 9.83 5.85 1.0 5.17 3.40 5.04 4.54 0.987 2.0 5.43 2.31 4.46 4.07 1.60 4.0 0.220 ND ND ND ND 8.0 BLOQ ND NDND ND Animal Weight (g) 0.250 0.256  0.249 0.252  0.004 Volume Dosed(mL) 1.58 1.61 1.57 1.59 0.02 C_(max) (ng/mL) 17.3 11.3 6.54 11.7 5.39t_(max) (hr) 0.250 0.250  0.250 0.250  0.000 t_(1/2) (hr) 0.600 0.9794.31 1.96 2.04 MRT_(last) (hr) 1.12 0.749  0.942 0.936  0.184 AUC_(last)(hr ng/mL) 22.4 9.10 10.3  14.0 7.37 AUC_(∞) (hr ng/mL) 22.6 12.4  ND³17.5 ND Dose-normalized Values¹ AUC_(last) (hr kg 2.24 0.910 1.03 1.40 0.737 ng/mL/mg) AUC_(∞) (hr kg ng/mL/mg) 2.26 1.24  ND³ 1.75 NDBioavailability (%)² 3.61 1.47 1.66 2.25 1.19 C_(max): maximum plasmaconcentration; t_(max): time of maximum plasma concentration; t_(1/2):half-life, data points used for half-life determination are in bold;MRT_(last): mean residence time, calculated to the last observable timepoint; AUC_(last): area under the curve, calculated to the lastobservable time point; AUC_(∞): area under the curve, extrapolated toinfinity; ND: not determined; BLOQ: below the limit of quantitation (2.5ng/mL); ¹Dose normalized by dividing the parameter by the nominal dosein mg/kg; ²Bioavailability determined by dividing the individualdose-normalized oral AUC_(last) values by the average IV AUC_(last)value 62.1 hr*ng/mL from Example 5; ³not determined because the AUC_(∞)was a greater than 25% extrapolation above the AUC_(last).

Following PO dosing of I-5 (Group 3), maximum plasma concentrations(average of 12.2±5.52 ng/mL) were observed between fifteen and thirtyminutes post dosing. Average half-life after PO dosing was 3.15 hours.Average exposure based on the dose normalized AUC_(last) was 2.38±1.47hr*kg*ng/mL/mg. Based on the IV data from Example 5, average oralbioavailability for I-5 was 3.84±2.37%. Results are shown in Table 7dand FIGS. 5E and 5F.

TABLE 7d Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of 1-5 at19.7 mg/kg in Male Sprague-Dawley Rats (Group 3). Oral (19 7 mg/kg I-5equals 10 mg/kg PEA Rat # Time (hr) 167 168 169 Mean SD 0 (pre-dose)3.66 BLOQ BLOQ ND ND 0.083 ND  3.92 6.53 5.23 ND 0.25 3.13 14.1 16.511.2 7.13 0.50 5.97 10.0 9.04 8.34 2.11 1.0 3.35 3.77 3.33 3.48  0.2482.0 3.43 4.84 8.43 5.57 2.58 4.0 ND  2.94 3.03 2.99 ND 8.0 BLOQ 1.461.69 1.58 ND Animal Weight (g)  0.255 0.256 0.251 0.254  0.003 VolumeDosed (mL) 1.68 1.69 1.66 1.68 0.02 C_(max) (ng/mL) 5.97 14.1 16.5 12.25.52 t_(max) (hr)  0.500 0.250 0.250 0.333  0.144 t_(1/2) (hr) ND³ 3.532.77 3.15 ND MRT_(last) (hr)  0.994 2.71 2.66 2.12  0.977 AUC_(last) (hrng/mL) 7.27 29.0 35.3 23.8 14.7  AUC_(∞) (hr ng/mL) ND³ 36.4 42.0 39.2ND Dose-normalized Values¹ AUC_(last) (hr kg  0.727 2.90 3.53 2.38 1.47ng/mL/mg) AUC_(∞) (hr kg ng/mL/mg) ND³ 3.64 4.20 3.92 ND Bioavailability(%)² 1.17 4.67 5.68 3.84 2.37 C_(max): maximum plasma concentration;t_(max): time of maximum plasma concentration; t_(1/2): half-life, datapoints used for half-life determination are in bold; MRT_(last): meanresidence time, calculated to the last observable time point;AUC_(last): area under the curve, calculated to the last observable timepoint; AUC_(∞): area under the curve, extrapolated to infinity; ND: notdetermined; BLOQ: below the limit of quantitation (2.5 ng/mL);¹Dose-normalized values determined by dividing the parameter by thenominal dose in mg/kg; ²Bioavailability determined by dividing theindividual dose-normalized oral AUC_(last) values by the average IVAUC_(last) value 62.1 hr*ng/mL from Example 5; ³not determined due tolack of quantifiable data points trailing the C_(max).

Following PO dosing of I-3 (Group 4), maximum plasma concentrations(average of 9.83±3.69 ng/mL) were observed between thirty minutes and 2hours post dosing. Average half-life was not determined; however, thehalf-life of one rat was 0.779 hours. Average exposure based on thedose-normalized AUC_(last) was 1.99±0.338 hr*kg*ng/mL/mg. Based on theIV data from Example 5, average oral bioavailability for I-3 was3.20±0.544%. Results are shown in Table 7e and FIGS. 5G and 5H.

TABLE 7e Individual and Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-3 at24.5 mg/kg in Male Sprague-Dawley Rats (Group 4). Oral (24.5 mg/kg I-3equals 10 mg/kg PEA) Rat # Time (hr) 170 171 172 Mean SD 0 (pre-dose)2.81 3.07 2.22 2.70  0.436 0.083  0.640 1.02 ND   0.830 ND 0.25 3.907.43 6.20 5.84 1.79 0.50 3.52 10.4 13.2  9.04 4.98 1.0 3.45 8.03 12.9 8.12 4.72 2.0 5.89 6.61 10.7  7.73 2.58 4.0 2.27 0.640 ND  1.46 ND 8.0ND  BLOQ ND  ND ND Animal Weight (g)  0.254 0.259  0.260  0.258  0.003Volume Dosed (mL) 2.08 2.12 2.13 2.11 0.03 C_(max) (ng/mL) 5.89 10.413.2  9.83 3.69 t_(max) (hr) 2.00 0.500  0.500 1.00  0.866 t_(1/2) (hr)ND³ 0.779 ND³ ND ND MRT_(last) (hr) 1.89 1.38 1.08 1.45  0.405AUC_(last) (hr ng/mL) 16.0  22.3 21.4  19.9  3.38 AUC_(∞) (hr ng/mL) ND³23.0 ND³ ND ND Dose-normalized Values¹ AUC_(last) (hr kg 1.60 2.23 2.141.99  0.338 ng/mL/mg) AUC_(∞) (hr kg ng/mL/mg) ND³ 2.30 ND³ ND NDBioavailability (%)² 2.58 3.59 3.44 3.20  0.544 C_(max): maximum plasmaconcentration; t_(max): time of maximum plasma concentration; t_(1/2):half-life, data points used for half-life determination are in bold;MRT_(last): mean residence time, calculated to the last observable timepoint; AUC_(last): area under the curve, calculated to the lastobservable time point; AUC_(∞): area under the curve, extrapolated toinfinity; ND: not determined; BLOQ: below the limit of quantitation (2.5ng/mL); ¹Dose-normalized by dividing the parameter by the nominal dosein mg/kg; ²Bioavailability determined by dividing the individualdose-normalized oral AUC_(last) values by the average IV AUC_(last)value 62.1 hr*ng/mL from Example 5; ³not determined due to lack ofquantifiable data points trailing the C_(max).

Following PO dosing of I-2 (Group 5), maximum plasma concentrations(average of 8.83±1.23 ng/mL) were observed between fifteen and thirtyminutes post dosing. Average half-life was not determined; however, thehalf-life of one rat was 0.797 hours. Average exposure based on thedose-normalized AUC_(last) was 0.854±0.164 hr*kg*ng/mL/mg. Based on theIV data from Example 5, average oral bioavailability for I-2 was1.38±0.264%. Results are shown in Table 7f and FIGS. 5I and 5J.

TABLE 7f Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-2 at12.5 mg/kg in Male Sprague-Dawley Rats (Group 5). Oral (12.5 mg/kg I-2equals 10 mg/kg PEA) Rat # Time (hr) 173 174 175 Mean SD 0 (pre-dose)2.67 1.41 1.73 1.94  0.655 0.083  0.220 3.07 2.02 1.77 1.44 0.25 7.7310.2 8.47 8.80 1.26 0.50 7.83 8.69 8.37 8.30  0.435 1.0 3.80 3.89 1.222.97 1.52 2.0 3.11 2.19 1.11 2.14 1.00 4.0 ND  BLOQ BLOQ ND ND 8.0 ND ND  BLOQ ND ND Animal Weight (g)  0.247 0.246  0.259  0.251  0.007Volume Dosed (mL) 1.04 1.04 1.09 1.06 0.03 C_(max) (ng/mL) 7.83 10.28.47 8.83 1.23 t_(max) (hr)  0.500 0.250  0.250  0.333  0.144 t_(1/2)(hr) ND³ 0.797 ND⁵ ND ND MRT_(last) (hr)  0.862 0.742  0.606  0.736 0.128 AUC_(last) (hr ng/mL) 9.09 9.84 6.70 8.54 1.64 AUC_(∞) (hr ng/mL)ND³ ND⁴ ND⁵ ND ND Dose-normalized Values¹ AUC_(last) (hr kg  0.909 0.984 0.670  0.854  0.164 ng/mL/mg) AUC_(∞) (hr kg ng/mL/mg) ND³ ND⁴ ND⁵ NDND Bioavailability (%)² 1.46 1.58 1.08 1.38  0.264 C_(max): maximumplasma concentration; t_(max): time of maximum plasma concentration;t_(1/2): half-life, data points used for half-life determination are inbold; MRT_(last): mean residence time, calculated to the last observabletime point; AUC_(last): area under the curve, calculated to the lastobservable time point; AUC_(∞): area under the curve, extrapolated toinfinity; ND: not determined; BLOQ: below the limit of quantitation (2.5ng/mL); ¹Dose-normalized by dividing the parameter by the nominal dosein mg/kg; ²Bioavailability determined by dividing the individualdose-normalized oral AUC_(last) values by the average IV AUC_(last)value 62.1 hr*ng/mL from Example 5; ³not determined due to lack ofquantifiable data points trailing the C_(max); ⁴not determined becausethe AUC_(∞) was a greater than 25% extrapolation above the AUC_(last);⁵not determined because the line defining the terminal elimination phasehad an r² <0.85.

Following PO dosing of I-11 (Group 6), maximum plasma concentrations(average of 15.5±3.01 ng/mL) were observed between fifteen minutes andthirty minutes post dosing. Average half-life was not determined;however, the half-life of one rat was 0.685 hours. Average exposurebased on the dose-normalized AUC_(last) was 2.79±0.808 hr*kg*ng/mL/mg.Based on the IV data from Example 5, average oral bioavailability forI-11 was 4.49±1.30%. Results are shown in Table 7g and FIGS. 5K and 5L.

TABLE 7g Individual Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-11 at20.7 mg/kg in Male Sprague-Dawley Rats (Group 6). Oral (20.7 mg/kg I-11equals 10 mg/kg PEA) Rat # Time (hr) 176 177 178 Mean SD 0 (pre-dose)1.62 1.89 BLOQ 1.76 ND 0.083 3.26 1.41 4.33 3.00 1.48 0.25 12.7 11.115.2 13.0 2.06 0.50 12.6 18.7 14.9 15.4 3.10 1.0 6.74 10.6 13.1 10.23.20 2.0 4.414 5.73 5.80 5.22  0.939 4.0 0.340 0.510 0.828 0.559  0.2488.0 BLOQ 0.520 2.62 1.57 ND Animal Weight (g) 0.258 0.252 0.259 0.256 0.004 Volume Dosed (mL) 1.78 1.74 1.79 1.77 0.03 C_(max) (ng/mL) 12.718.7 15.2 15.5 3.01 t_(max) (hr) 0.250 0.500 0.250 0.333  0.144 t_(1/2)(hr) 0.685 ND³ ND³ ND ND MRT_(last) (hr) 1.13 1.52 2.33 1.66  0.612AUC_(last) (hr ng/mL) 19.5 28.7 35.5 27.9 8.08 AUC_(∞) (hr ng/mL) 19.8ND³ ND³ ND ND Dose-normalized Values¹ AUC_(last) (hr kg 1.95 2.87 3.552.79  0.808 ng/mL/mg) AUC_(∞) (hr kg ng/mL/mg) 1.98 ND³ ND³ ND NDBioavailability (%)² 3.13 4.62 5.73 4.49 1.30 C_(max): maximum plasmaconcentration; t_(max): time of maximum plasma concentration; t_(1/2):half-life, data points used for half-life determination are in bold;MRT_(last): mean residence time, calculated to the last observable timepoint; AUC_(last): area under the curve, calculated to the lastobservable time point; AUC_(∞): area under the curve, extrapolated toinfinity; ND: not determined; BLOQ: below the limit of quantitation (2.5ng/mL); ¹Dose-normalized by dividing the parameter by the nominal dosein mg/kg; ²Bioavailability determined by dividing the individualdose-normalized oral AUC_(last) values by the average IV AUC_(last)value 62.1 hr*ng/mL from Example 5; ³not determined because the linedefining the terminal elimination phase had an r² < 0.85.

Following PO dosing of I-7 (Group 7), maximum plasma concentrations(average of 3.52 ng/mL) were observed between zero and fifteen minuteshour post dosing. Average half-life was not determined; however, thehalf-life of one rat was 0.736 hours. Average exposure based on thedose-normalized AUC_(last) was 1.23 hr*kg*ng/mL/mg. Based on the IV datafrom Example 5, average oral bioavailability for I-7 was 1.98%. Resultsare shown in Table 7h and FIGS. 5M and 5N.

TABLE 7h Individual and Average Plasma Concentrations (mg/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-7 at24.5 mg/kg in Male Sprague-Dawley Rats (Group 7). Oral (24.5 mg/kg I-7equals 10 mg/kg PEA) Rat # Time (hr) 179 180 181 Mean SD 0 (pre-dose)3.15 BLOQ 3.85 3.50 ND 0.083 ND 2.88 ND ND ND 0.25 1.65 3.89 ND 2.77 ND0.50 1.57 3.28 ND 2.43 ND 1.0 1.23 2.36 ND 1.80 ND 2.0 0.400 3.43 ND1.92 ND 4.0 ND 2.46 ND ND ND 8.0 ND 2.85 ND ND ND Animal Weight (kg)0.254  0.249  0.263  0.255  0.007 Volume Dosed (mL) 2.08 2.04 2.16 2.090.06 C_(max) (ng/mL) 3.15 3.89 ND 3.52 ND t_(max) (hr) 0.000  0.250 ND 0.125 ND t_(1/2) (hr) 0.736 ND³ ND ND ND MRT_(last) (hr) 0.779 3.93 ND2.35 ND AUC_(last) (hr ng/mL) 2.19 22.4  ND 12.3  ND AUC_(∞) (hr ng/mL)2.61 ND³ ND ND ND Dose-normalized Values¹ ND AUC_(last) (hr kg ng/mL/mg)0.219 2.24 ND 1.23 ND AUC_(∞) (hr kg ng/mL/mg) 0.261 ND³ ND ND NDBioavailability (%)² 0.352 3.61 ND 1.98 ND C_(max): maximum plasmaconcentration; t_(max): time of maximum plasma concentration; t_(1/2):half-life, data points used for half-life determination are in bold;MRT_(last): mean residence time, calculated to the last observable timepoint; AUC_(last): area under the curve, calculated to the lastobservable time point; AUC_(∞): area under the curve, extrapolated toinfinity; ND: not determined; BLOQ: below the limit of quantitation (2.5ng/mL); ¹Dose-normalized by dividing the parameter by the nominal dosein mg/kg; ²Bioavailability determined by dividing the individualdose-normalized oral AUC_(last) values by the average IV AUC_(last)value 62.1 hr*ng/mL from Example 5; ³not determined because the linedefining the terminal elimination phase had an r² <0.85.

Example 8: PEA Stability in Human, Rat Mouse and Dog Liver Microsomes,Human, Rat, Mouse and Dog Liver S9 Fraction, Human, Rat, Mouse and DogIntestinal S9 Fraction, Human, Rat, Mouse and Dog Plasma, and SimulatedIntestinal Fluid

The present Example describes PEA stability observed in 1) human, rat,mouse, and dog liver microsomes; 2) human, rat, mouse, dog liver S9fraction; human, rat, mouse, and dog intestinal S9 fraction; 4) human,rat, mouse, and dog plasma; and 5) simulated intestinal fluid containingvarious enzymes.

Liver Microsomal Stability

Mixed-gender human (Lot #1210347), male Sprague-Dawley rat (Lot#1310030), male CD-I mouse (Lot #1510043), and male Beagle dog (Lot#0810143) liver microsomes were provided. Reaction mixture, minuscofactors, was prepared as described below. Test article was added intothe reaction mixture at a final concentration of 1 μM. Control compound,testosterone, was run simultaneously with the test article in a separatereaction. An aliquot of the reaction mixture (without cofactor) wasequilibrated in a shaking water bath at 37° C. for 3 minutes. Thereaction was initiated by the addition of cofactor, and the mixture wasincubated in a shaking water bath at 37° C. Aliquots (100 μL) werewithdrawn at 0, 10, 20, 30, and 60 minutes. Test article samples wereimmediately combined with 300 μL of ice-cold acetonitrile containing 1%formic acid. Control samples were immediately combined with 400 μL ofice-cold 50/50 acetonitrile (ACN)/dH₂O containing 0.1% formic acid andinternal standard to terminate the reaction. Samples were then mixed andcentrifuged to precipitate proteins. Calibration standards were preparedin matched matrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both dosed prodrug and expected drug (PEA).Analytical conditions are outlined in Appendix 8-1. Test articleconcentration at each time point was compared to test articleconcentration at time 0 to determine the percent remaining at each timepoint. Half-lives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 8aand 8b.

Reaction Composition

Liver Microsomes 0.5 mg/mL NADPH (cofactor) 1 mM Potassium Phosphate pH7.4 100 mM Magnesium Chloride 5 mM Test Article 1 μL

TABLE 8a PEA stability observed in human, rat, mouse, and dog livermicrosomes. CL_(int) ^(b) % Remaining of Initial (n = 1) Half- (mL/min/Test 10 20 30 60 life^(a) mg Article Species 0 min min min min min (min)protein) I-6 Human 100 82 58 66 68 >60 (97) <0.0231 (0.0143) Rat 100 6867 62 53 >60 (63) <0.0231 (0.0219) Mouse 100 84 74 73 61 >60 (84)<0.0231 (0.0164) Dog 100 103 100 91 77 >60 <0.0231 I-2 Human 100 10 1.6<1.0 <1.0 3.1 0.451 Rat 100 <1.0 <1.0 <1.0 <1.0 <1.0 >1.38 Mouse 100<1.0 <1.0 <1.0 <1.0 <1.0 >1.38 Dog 100 5.4 <1.0 <1.0 <1.0 2.4 0.581 I-9Human 100 <1.0 <1.0 <1.0 <1.0 <1.0 >1.38 Rat 100 1.9 <1.0 <1.0 <1.0 1.80.791 Mouse 100 <1.0 <1.0 <1.0 <1.0 <1.0 >1.38 Dog 100 <1.0 <1.0 <1.0<1.0 <1.0 >1.38 I-3 Human 100 83 79 74 53 >60 (69) <0.0231 (0.0200) Rat100 81 88 72 68  >60 (112) <0.0231 (0.0124) Mouse 100 82 83 60 70  >60(100) <0.0231 (0.0138) Dog 100 94 85 85 74 >60 <0.0231 I-5 Human 100 9082 79 62 >60 (90) <0.0231 (0.0154) Rat 100 77 70 69 57 >60 (75) <0.0231(0.0186) Mouse 100 72 57 56 55 59 0.0236 Dog 100 84 75 75 67  >60 (104)<0.0231 (0.0133) I-11 Human 100 3.1 <1.0 <1.0 <1.0 2.0 0.696 Rat 100 3.3<1.0 <1.0 <1.0 2.0 0.681 Mouse 100 3.2 <1.0 <1.0 <1.0 2.0 0.687 Dog 1003.6 <1.0 <1.0 <1.0 2.1 0.665 I-7 Human 100 4.6 1.3 <1.0 <1.0 2.3 0.612Rat 100 45 17 11 2.1 8.5 0.164 Mouse 100 1.7 1.2 <1.0 <1.0 1.7 0.814 Dog100 1.6 1.4 1.3 <1.0 1.7 0.823 ^(a)When the calculated half-life islonger than the duration of the experiment, the half-life is expressedas > the longest incubation time. Similarly, if the calculated half-lifeis less than the shortest time point, the half-life is expressed as <that time point and the calculated half-life is also listed inparentheses. ^(b)Intrinsic clearance (CL_(int)) was calculated based onCL_(int) = k/P, where k is the elimination rate constant and P is theprotein concentration in the incubation.

Half- CL_(int) Acceptable Control life (ml/min/mg Range Compound Species(min) protein) (t_(1/2), min) Testosterone Human 27 0.0505 ≤41 Rat 1.80.760 ≤15 Mouse 4.9 0.285 ≤15 Dog 33 0.0415 ≤40

TABLE 8b Measured Concentrations of Drug. Dosed Concentration (μM) Test10 20 30 60 Article Species Analyte 0 min min min min min I-6 Human PEA0 0 0 0 0 Rat 0.14 0.064 0.017 0.0098 0.0012 Mouse 0.022 0.055 0.0390.041 0.027 Dog 0.030 0.087 0.091 0.080 0.084 I-2 Human 0.15 0.23 0.190.15 0.071 Rat 0.40 0.11 0.028 0.0083 0 Mouse 0.48 0.34 0.19 0.12 0.023Dog 0.23 0.48 0.38 0.29 0.17 I-9 Human 0.47 0.39 0.22 0.16 0.058 Rat0.22 0.066 0.017 0 0 Mouse 0.52 0.29 0.18 0.11 0.036 Dog 0.39 0.42 0.320.26 0.18 I-3 Human 0 0.026 0.017 0.019 0.027 Rat 0.032 0.039 0.0180.0067 0 Mouse 0.0079 0.025 0.025 0.042 0.025 Dog 0.00074 0.023 0.0370.042 0.053 I-5 Human 0.014 0.020 0.025 0.022 0.016 Rat 0.034 0.0330.015 0.015 0.0027 Mouse 0.048 0.080 0.052 0.048 0.024 Dog 0.025 0.0810.090 0.087 0.063 I-11 Human 0.046 0.29 0.20 0.16 0.057 Rat 0.023 0.0480.031 0.0086 0 Mouse 0.22 0.32 0.18 0.12 0.030 Dog 0.032 0.32 0.26 0.200.097 I-7 Human 0.22 0.39 0.28 0.22 0.080 Rat 0.054 0.14 0.087 0.0450.0085 Mouse 0.28 0.29 0.18 0.11 0.034 Dog 0.59 0.50 0.47 0.38 0.20

Liver S9 Stability

Mixed gender human (Lot #1210091), male Sprague-Dawley rat (Lot#1410265), male CD-1 mouse (Lot #1510255), and male Beagle dog (Lot#1210278) liver S9 fraction were provided. Reaction mixture, minuscofactors, was prepared as described below. Test article was added intothe reaction mixture at a final concentration of 1 μM. Controlcompounds, testosterone and 7-hydroxycoumarin (7-HC), were runsimultaneously with the test article in a separate reaction. An aliquotof the reaction mixture (without cofactor cocktail) was equilibrated ina shaking water bath at 37° C. for 3 minutes. The reaction was initiatedby the addition of cofactor cocktail (see below), and the mixture wasthen incubated in a shaking water bath at 37° C. Aliquots (100 μL) werewithdrawn 0, 10, 20, 30 and 60 minutes. Test article samples wereimmediately combined with 300 μL of ice-cold acetonitrile containing 1%formic acid. Control samples were immediately combined with 400 μL ofice-cold 50/50 acetonitrile (ACN)/dH₂O containing 0.1% formic acid andinternal standard to terminate the reaction. Samples were then mixed andcentrifuged to precipitate proteins. Calibration standards were preparedin matched matrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both the dosed prodrug and the expected drug(PEA). Analytical conditions are outlined in Appendix 8-1. Test articleconcentration at each time point was compared to the test articleconcentration at time 0 to determine the percent remaining at each timepoint. Half-lives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 8cand 8d.

Reaction Composition

Liver S9 Fraction 1.0 mg/mL NADPH (cofactor) 1 mM UDPGA (cofactor) 1 mMPAPS (cofactor) 1 mM GSH (cofactor) 1 mM Potassium Phosphate pH 7.4 100mM Magnesium Chloride 5 mM Test Article 1 μM

TABLE 8c PEA stability observed in human, rat, mouse, and dog liver S9.CL_(int) ^(b) % Remaining of Initial (n = 1) Half- (mL/min/ Test 10 2030 60 life^(a) mg Article Species 0 min min min min min (min) protein)I-6 Human 100 80 102 95 77 >60 <0.0116 Rat 100 64 82 93 89 >60 <0.0116Mouse 100 87 87 99 75 >60 <0.0116 Dog 100 101 106 102 110 >60 <0.0116I-2 Human 100 6.0 1.3 <1.0 <1.0 2.5 0.279 Rat 100 <1.0 <1.0 <1.0 <1.0<1.0 >0.691 Mouse 100 <1.0 <1.0 <1.0 <1.0 <1.0 >0.691 Dog 100 5.1 2.0<1.0 <1.0 2.4 0.293 I-9 Human 100 <1.0 <1.0 <1.0 <1.0 <1.0 >0.691 Rat100 13 2.1 <1.0 <1.0 3.4 0.207 Mouse 100 <1.0 <1.0 <1.0 <1.0 <1.0 >0.691Dog 100 <1.0 <1.0 <1.0 <1.0 <1.0 >0.691 I-3 Human 100 104 89 95 66  >60(104) <0.0116 (0.00665) Rat 100 66 69 71 52 >60 (72) <0.0116 (0.00967)Mouse 100 82 61 78 61 >60 (87) <0.0116 (0.00797) Dog 100 104 89 8086 >60 <0.0116 I-5 Human 100 80 81 70 72 >60 <0.0116 Rat 100 98 82 7567 >60 (91) <0.0116 (0.00764) Mouse 100 66 50 59 50 53 0.0132 Dog 100 7279 74 85 >60 <0.0116 I-11 Human 100 2.8 <1.0 <1.0 <1.0 1.9 0.359 Rat 100<1.0 <1.0 <1.0 <1.0 <1.0 >0.691 Mouse 100 <1.0 <1.0 <1.0 <1.0<1.0 >0.691 Dog 100 4.6 1.6 <1.0 <1.0 2.3 0.306 I-7 Human 100 1.9 <1.0<1.0 <1.0 1.8 0.396 Rat 100 43 22 11 1.4 8.8 0.0786 Mouse 100 1.6 <1.0<1.0 <1.0 1.7 0.415 Dog 100 <1.0 <1.0 <1.0 <1.0 <1.0 >0.691 ^(a)When thecalculated half-life is longer than the duration of the experiment, thehalf-life is expressed as > the longest incubation time. Similarly, ifthe calculated half-life is less than the shortest time point, thehalf-life is expressed as < that time point and the calculated half-lifeis also listed in parentheses. ^(b)Intrinsic clearance (CL_(int)) wascalculated based on CL_(int) = k/P, where k is the elimination rateconstant and P is the protein concentration in the incubation.

Half- CL_(int) Acceptable Control life (ml/min/mg Range Compound Species(min) protein) (t_(1/2), min) Testosterone Human 16 0.0447 ≤34 Rat 4.70.148 ≤15 Mouse 8.7 0.0800 ≤37 Dog 14 0.0485 ≤42 7-hydroxycoumarin Human10 0.0668 ≤18 Rat 4.6 0.152 ≤15 Mouse 4.1 0.171 ≤15 Dog 1.4 0.502 ≤15

TABLE 8d Measured Concentrations of Drug. Dosed Concentration (μM) Test10 20 30 60 Article Species Analyte 0 min min min min min I-6 Human PEA0.014 0.037 0.022 0.022 0.016 Rat 0.020 0.035 0.021 0.021 0.013 Mouse0.030 0.041 0.033 0.031 0.015 Dog 0.016 0.044 0.041 0.049 0.037 I-2Human 0.20 0.23 0.18 0.096 0.033 Rat 0.50 0.31 0.17 0.11 0.023 Mouse0.51 0.26 0.19 0.12 0.022 Dog 0.23 0.38 0.35 0.21 0.11 I-9 Human 0.350.30 0.18 0.088 0.028 Rat 0.20 0.31 0.18 0.11 0.029 Mouse 0.52 0.23 0.130.077 0.022 Dog 0.32 0.29 0.21 0.18 0.078 I-3 Human 0.00032 0.0058 0.0170.015 0.0096 Rat 0.015 0.045 0.033 0.017 0.013 Mouse 0.013 0.031 0.0220.023 0.025 Dog 0.010 0.030 0.035 0.031 0.029 I-5 Human 0.024 0.0360.036 0.025 0.018 Rat 0.020 0.025 0.037 0.026 0.013 Mouse 0.046 0.0610.043 0.029 0.018 Dog 0.024 0.044 0.036 0.041 0.038 I-11 Human 0.0470.25 0.18 0.14 0.033 Rat 0 0.14 0.091 0.059 0.0062 Mouse 0.18 0.24 0.130.083 0.015 Dog 0.069 0.51 0.34 0.29 0.13 I-7 Human 0.20 0.32 0.17 0.130.042 Rat 0.045 0.15 0.16 0.12 0.039 Mouse 0.22 0.22 0.14 0.084 0.020Dog 0.21 0.16 0.15 0.12 0.049

Intestinal S9 Fraction Stability

Mixed-gender human (Lot #1410073), male Sprague-Dawley rat (Lot#1510303), male CD-1 mouse (Lot #1510194), and male Beagle dog (Lot#1510226) intestinal S9 fraction were provided. Reaction mixture, minuscofactors, was prepared as described below. Test article was added intothe reaction mixture at a final concentration of 1 μM. Controlcompounds, testosterone and 7-hydroxycoumarin, were run simultaneouslywith the test article in a separate reaction. An aliquot of the reactionmixture (without cofactor cocktail) was equilibrated in a shaking waterbath at 37° C. for 3 minutes. The reaction was initiated by the additionof cofactor cocktail, and the mixture was incubated in a shaking waterbath at 37° C. Aliquots (100 μL) were withdrawn at 0, 10, 20, 30, and 60minutes. Test article samples were immediately combined with 300 μL ofice-cold acetonitrile containing 1% formic acid. Control samples wereimmediately combined with 400 μL of ice-cold 50/50 acetonitrile(ACN)/dH₂O containing 0.1% formic acid and internal standard toterminate the reaction. Samples were then mixed and centrifuged toprecipitate proteins. Calibration standards were prepared in matchedmatrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both the dosed prodrug and the expected drug(PEA). Analytical conditions are outlined in Appendix 8-1. Test articleconcentration at each time point was compared to test articleconcentration at time 0 to determine the percent remaining at each timepoint. Halflives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 8eand 8f.

Reaction Composition

Intestinal S9 Fraction 1.0 mg/mL NADPH (cofactor) 1 mM UDPGA (cofactor)1 mM PAPS (cofactor) 1 mM GSH (cofactor) 1 mM Potassium Phosphate, pH7.4 100 mM Magnesium Chloride 5 mM Test Article 1 μM

TABLE 8e PEA stability observed in human, rat, mouse, and dog intestinalS9 fraction. CL_(int) ^(b) % Remaining of Initial (n = 1) Half- (mL/Test 10 20 30 60 life^(a) min/mg Article Species 0 min min min min min(min) protein) I-6 Human 100 109 103 82 70  >60 (102) <0.0116 (0.00682)Rat 100 78 92 79 59 >60 (91) <0.0116 (0.00765) Mouse 100 62 69 61 53 >60(66) <0.0116 (0.0105)  Dog 100 54 53 45 38 34 0.0201 I-2 Human 100 73 4128 9.6 17 0.0415 Rat 100 35 <1.0 <1.0 <1.0 5.6 0.123 Mouse 100 <1.0 <1.0<1.0 <1.0 <1.0 >0.691 Dog 100 36 12 4.2 <1.0 6.7 0.104 I-9 Human 100 285.7 1.2 <1.0 5.3 0.131 Rat 100 45 26 15 3.6 10 0.0695 Mouse 100 56 33 216.5 13 0.0537 Dog 100 <1.0 <1.0 <1.0 <1.0 <1.0 >0.691 I-3 Human 100 7686 83 60 >60 (98) <0.0116 (0.00706) Rat 100 53 46 42 31 26 0.0268 Mouse100 68 59 49 33 33 0.0211 Dog 100 46 46 34 18 19 0.0368 I-5 Human 100 8249 38 45 32 0.0216 Rat 100 57 48 46 44 38 0.0182 Mouse 100 68 67 66 4960 0.0116 Dog 100 50 48 36 36 26 0.0263 I-11 Human 100 4.1 1.2 <1.0 <1.02.2 0.318 Rat 100 1.4 <1.0 <1.0 <1.0 1.6 0.425 Mouse 100 <1.0 <1.0 <1.0<1.0 <1.0 >0.691 Dog 100 1.8 <1.0 <1.0 <1.0 1.7 0.404 I-7 Human 100 4622 9.8 3.8 9.0 0.0772 Rat 100 60 37 31 13 16 0.0431 Mouse 100 59 35 275.7 14 0.0485 Dog 100 1.1 <1.0 <1.0 <1.0 1.5 0.452 ^(a)When thecalculated half-life is longer than the duration of the experiment, thehalf-life is expressed as > the longest incubation time. Similarly, ifthe calculated half-life is less than the shortest time point, thehalf-life is expressed as < that time point and the calculated half-lifeis also listed in parentheses. ^(b)Intrinsic clearance (CL_(int)) wascalculated based on CL_(int) = k/P, where k is the elimination rateconstant and P is the protein concentration in the incubation.

CL_(int) Control Half-life (ml/min/mg Compound Species (min) protein)Testosterone Human 7.8 0.0889 Rat >60 (106) <0.0116 (0.00652) Mouse >60(75)  <0.0116 (0.00919) Dog >60 (96)  <0.0116 (0.00722)7-hydroxycoumarin Human 13 0.0545 Rat 27 0.0257 Mouse 5.0 0.139 Dog 8.60.0803

TABLE 8f Measured Concentrations of Drug. Dosed Concentration (μM) Test10 20 30 60 Article Species Analyte 0 min min min min min I-6 Human PEA0.0089 0.023 0.043 0.056 0.072 Rat 0.069 0.088 0.094 0.10 0.11 Mouse0.12 0.14 0.17 0.15 0.17 Dog 0.26 0.42 0.38 0.35 0.36 I-2 Human 0.0110.057 0.075 0.067 0.11 Rat 0.69 0.63 0.60 0.52 0.52 Mouse 0.52 0.50 0.520.45 0.45 Dog 0.11 0.40 0.50 0.53 0.54 I-9 Human 0.048 0.18 0.23 0.220.18 Rat 0.034 0.22 0.31 0.36 0.39 Mouse 0.0024 0.12 0.15 0.19 0.23 Dog0.26 0.48 0.47 0.46 0.45 I-3 Human 0 0.0027 0.025 0.034 0.048 Rat 00.024 0.037 0.047 0.073 Mouse 0 0.026 0.024 0.026 0.043 Dog 0.015 0.230.28 0.29 0.50 I-5 Human 0 0.050 0.063 0.074 0.10 Rat 0.029 0.062 0.0990.11 0.18 Mouse 0.050 0.11 0.076 0.093 0.14 Dog 0.13 0.26 0.25 0.25 0.28I-11 Human 0.018 0.13 0.20 0.21 0.18 Rat 0 0.13 0.24 0.35 0.38 Mouse 00.021 0.059 0.12 0.084 Dog 0.032 0.23 0.38 0.74 0.88 I-7 Human 0.0900.46 0.62 0.63 0.48 Rat 0.042 0.24 0.35 0.47 0.60 Mouse 0.033 0.24 0.290.39 0.38 Dog 0.38 0.36 0.55 0.51 0.50

Plasma Stability

Studies were carried out in mixed-gender human plasma (Lot #GLP530-5),male Sprague-Dawley rat (Lot #RAT297944, RAT313140), male CD-1 mouse(Lot #MSE237700), and male Beagle dog (Lot #BGL87670, BGL82614),collected on sodium heparin. Plasma was adjusted to pH 7.4 prior toinitiating the experiments. DMSO stocks were first prepared for the testarticles. Aliquots of the DMSO solutions were dosed into 700 μL ofplasma, which had been pre-warmed to 37° C., at a final test articleconcentration of 1 μM. Aliquots (100 μL) were taken at each time point(0, 15, 30, 60, and 120 minutes) and were immediately combined with 300μL of ice-cold acetonitrile containing 1% formic acid. Samples werestored at 4° C. until the end of the experiment. After the final timepoint was sampled, the plate was mixed and then centrifuged at 3,000 rpmfor 10 minutes. Calibration standards were prepared in matched matrix.Samples and standards were assayed by LC-MS/MS using electrosprayionization for both the dosed prodrug and the expected drug (PEA).Analytical conditions are outlined in Appendix 8-1. Test articleconcentration at each time point was compared to test articleconcentration at time 0 to determine the percent remaining at each timepoint. Half-lives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 8gand 8h.

TABLE 8g PEA Stability observed in human, rat, mouse, and dog plasma. %Remaining of Initial (n = 1) Half- Test 15 30 60 120 life^(a) ArticleSpecies 0 min min min min min (min) I-6 Human 100 79 102 74 53 >120(147) Rat 100 93 119 100 29 110 Mouse 100 83 89 39 23 56 Dog 100 55 4234 22 36 I-2 Human 100 96 91 84 51 >120 (135) Rat 100 58 59 46 18 49Mouse 100 72 71 56 21 62 Dog 100 95 71 54 24 61 I-9 Human 100 6.4 1.4<1.0 <1.0 3.8 Rat 100 2.5 <1.0 <1.0 <1.0 2.8 Mouse 100 49 17 2.3 <1.0 13Dog 100 8.7 12 1.2 <1.0 4.7 I-3 Human 100 109 84 65 77 >120 (211) Rat100 94 79 76 79 >120 Mouse 100 23 24 9.1 4.7 9.4 Dog 100 90 90 9599 >120 I-5 Human 100 110 81 82 69 >120 (195) Rat 100 85 90 87 83 >120Mouse 100 109 86 73 75 >120 (213) Dog 100 87 98 87 83 >120 I-11 Human100 67 47 34 10 34 Rat 100 9.0 23 <1.0 <1.0 5.6 Mouse 100 2.9 2.1 1.9<1.0 3.0 Dog 100 61 40 18 5.4 23 I-7 Human 100 <1.0 <1.0 <1.0 <1.0 <1.0Rat 100 <1.0 <1.0 <1.0 <1.0 <1.0 Mouse 100 <1.0 <1.0 <1.0 <1.0 <1.0 Dog100 <1.0 <1.0 <1.0 <1.0 <1.0

TABLE 8h Measured Concentrations of Drug. Dosed Concentration (μM) Test15 30 60 120 Article Species Analyte 0 min min min min min I-6 Human PEA0 0 0 0 0 Rat 0 0 0.020 0.017 0.056 Mouse 0 0 0.015 0.059 0.11 Dog 0 0 00 0 I-2 Human 0.00043 0.035 0.064 0.10 0.21 Rat 0.052 0.22 0.28 0.280.38 Mouse 0.019 0.068 0.095 0.14 0.23 Dog 0.0061 0.12 0.18 0.21 0.26I-9 Human 0.049 0.41 0.37 0.49 0.35 Rat 0.77 1.2 1.3 1.2 1.1 Mouse 0.510.60 0.62 0.64 0.89 Dog 0.13 0.58 0.61 0.62 0.55 I-3 Human 0 0 0 00.0071 Rat 0.0029 0.0043 0.013 0.020 0.033 Mouse 0.015 0.10 0.25 0.360.42 Dog 0 0 0 0 0 I-5 Human 0.0017 0.034 0.055 0.083 0.096 Rat 0.0210.11 0.14 0.18 0.17 Mouse 0.074 0.19 0.19 0.18 0.29 Dog 0 0.040 0.0470.070 0.072 I-11 Human 0.011 0.13 0.26 0.38 0.53 Rat 0.0022 0.016 0.0170.025 0.025 Mouse 0.19 0.38 0.56 0.60 0.76 Dog 0.012 0.20 0.35 0.52 0.55I-7 Human 0.11 0.54 0.53 0.49 0.53 Rat 0.29 0.59 0.54 0.56 0.60 Mouse0.87 0.75 0.81 0.81 0.91 Dog 0.17 0.52 0.55 0.53 0.51

Simulated Intestinal Fluid Stability

Studies were carried out in simulated intestinal fluid in the presenceof various enzymes. Simulated intestinal fluid was prepared bydissolving 6.8 g of monobasic potassium phosphate in 1.0 L of water.Aliquots of this solution were taken and the pH was adjusted to 6.8.Individual enzymes were then spiked into aliquots for each experiment. ADMSO stock was first prepared for the test article. Aliquots of the DMSOsolution were dosed into 700 μL of matrix, which had been pre-warmed to37° C., at a final test article concentration of 1 μM. Aliquots (100 μL)were taken at each time point (0, 15, 30, 60, and 120 minutes) and wereimmediately combined with 300 μL of ice-cold acetonitrile containing 1%formic acid. Samples were stored at 4° C. until the end of theexperiment. After the final time point was sampled, the plate was mixedand then centrifuged at 3,000 rpm for 10 minutes. Calibration standardswere prepared in matched matrix. Samples and standards were assayed byLC-MS/MS using electrospray ionization for both the dosed prodrug andthe expected drug (PEA). Analytical conditions are outlined in Appendix8-1. Test article concentration at each time point was compared to testarticle concentration at time 0 to determine the percent remaining ateach time point. Half-lives were calculated using GraphPad software,fitting to a single-phase exponential decay equation. Results are shownin Tables 8i and 8j.

TABLE 8i PEA stability observed in simulated intestinal fluid (SIF). %Remaining of Initial (n = 1) Half- Test 15 30 60 120 life^(a) ArticleTreatment 0 min min min min min (min) I-6 SIF + Pancreatin 100 27 11 3.11.7 8.3 SIF + Elastase 100 95 74 64 69 >120 (185) SIF + CarboxypeptidaseA 100 77 46 60 56 >120 (134) SIF + Carboxypeptidase B 100 87 93 54 19 62SIF + Chymotrypsin 100 106 99 51 55 101 SIF + Trypsin 100 116 87 83123 >120 I-2 SIF + Pancreatin 100 93 91 98 60 >120 (205) SIF + Elastase100 61 42 30 28 37 SIF + Carboxypeptidase A 100 79 70 56 43 92 SIF +Carboxypeptidase B 100 75 54 40 17 43 SIF + Chymotrypsin 100 78 70 6058 >120 (144) SIF + Trypsin 100 82 72 75 46 >120 (121) I-9 SIF +Pancreatin 100 35 34 20 13 17 SIF + Elastase 100 49 22 10 6.3 15 SIF +Carboxypeptidase A 100 43 15 10 5.5 12 SIF + Carboxypeptidase B 100 4414 6.1 4.7 12 SIF + Chymotrypsin 100 45 24 11 7.2 14 SIF + Trypsin 10067 49 22 12 29 I-3 SIF + Pancreatin 100 <1.0 <1.0 <1.0 <1.0 <1.0 SIF +Elastase 100 94 61 44 29 55 SIF + Carboxypeptidase A 100 76 66 36 20 46SIF + Carboxypeptidase B 100 86 77 41 15 49 SIF + Chymotrypsin 100 10284 61 53 107 SIF + Trypsin 100 67 60 55 43 93 I-5 SIF + Pancreatin 10013 <1.0 <1.0 <1.0 5.1 SIF + Elastase 100 103 70 48 35 65 SIF +Carboxypeptidase A 100 84 63 39 15 44 SIF + Carboxypeptidase B 100 94 6243 14 45 SIF + Chymotrypsin 100 75 59 37 23 47 SIF + Trypsin 100 81 6744 26 56 I-11 SIF + Pancreatin 100 <1.0 <1.0 <1.0 <1.0 <1.0 SIF +Elastase 100 70 50 33 17 37 SIF + Carboxypeptidase A 100 73 78 42 18 53SIF + Carboxypeptidase B 100 68 50 33 9.6 34 SIF + Chymotrypsin 100 8463 42 21 50 SIF + Trypsin 100 77 62 45 23 53 I-7 SIF + Pancreatin 100 4019 6.5 2.7 12 SIF + Elastase 100 44 14 3.8 3.5 12 SIF + CarboxypeptidaseA 100 30 10 4.1 3.9 8.8 SIF + Carboxypeptidase B 100 33 14 5.7 2.2 10SIF + Chymotrypsin 100 42 23 13 15 14 SIF + Trypsin 100 66 44 38 33 50

TABLE 8j Measured Concentrations of Drug. Test Concentration (μM)Article Treatment Analyte 0 min 15 min 30 min 60 min 120 min I-6 SIF +Pancreatin PEA — — — — — SIF + Elastase 0 0 0 0 0 SIF + CarboxypeptidaseA 0 0 0 0 0 SIF + Carboxypeptidase B 0 0 0 0 0 SIF + Chymotrypsin 0 0 00 0 SIF + Trypsin 0 0 0 0 0 I-2 SIF + Pancreatin — — — — — SIF +Elastase 0 0 0 0 0 SIF + Carboxypeptidase A 0 0 0 0 0 SIF +Carboxypeptidase B 0 0 0 0 0 SIF + Chymotrypsin 0 0 0 0 0 SIF + Trypsin0 0 0 0 0 I-9 SIF + Pancreatin — — — — — SIF + Elastase 0 0 0 0 0 SIF +Carboxypeptidase A 0 0 0 0 0 SIF + Carboxypeptidase B 0 0 0 0 0 SIF +Chymotrypsin 0 0 0 0 0 SIF + Trypsin 0 0 0 0 0 I-3 SIF + Pancreatin — —— — — SIF + Elastase 0 0 0 0 0 SIF + Carboxypeptidase A 0 0 0 0 0 SIF +Carboxypeptidase B 0 0 0 0 0 SIF + Chymotrypsin 0 0 0 0 0 SIF + Trypsin0 0 0 0 0 I-5 SIF + Pancreatin — — — — — SIF + Elastase 0 0 0 0 0 SIF +Carboxypeptidase A 0 0 0 0 0 SIF + Carboxypeptidase B 0 0 0 0 0 SIF +Chymotrypsin 0 0 0 0 0 SIF + Trypsin 0 0 0 0 0 I-11 SIF + Pancreatin — —— — — SIF + Elastase 0 0 0 0 0 SIF + Carboxypeptidase A 0 0 0 0 0 SIF +Carboxypeptidase B 0 0 0 0 0 SIF + Chymotrypsin 0 0 0 0 0 SIF + Trypsin0 0 0 0 0 I-7 SIF + Pancreatin — — — — — SIF + Elastase 0 0 0 0 0 SIF +Carboxypeptidase A 0 0 0 0 0 SIF + Carboxypeptidase B 0 0 0 0 0 SIF +Chymotrypsin 0 0 0 0 0 SIF + Trypsin 0 0 0 0 0 PEA was found to beendogenous in pancreatin and thus was not quantified in the assaysamples.

Appendix 8-1 Liquid Chromatography

Column: Waters ACQUITY UPLC BEH C18 30 × 2.1 mm, 1.7 μm M.P. Buffer: 25mM ammonium formate buffer,, pH 3.5 Aqueous Reservoir (A): 90% water,10% buffer Organic Reservoir (B): 90% acetonitrile, 10% buffer FlowRate: 0.8 mL/minute Gradient Program:

TABLE 8-1 Liquid Chromatography Gradient Program Time (mm) % A % B 0.0050 50 0.75 1 99 1.25 1 99 1.30 50 50 1.50 50 50 Total Run Time: 1.5minutes Autosampler: 5 μL injection volume Wash1:water/methanol/2-propanol: 1/1/1; with 0.2% formic acid Wash2: 0.1%formic acid in water Mass Spectrometer Instrument: PE SCIEX API 4000Interface: Turbo Ionspray Mode: Multiple reaction monitoring Method: 1.5minute duration Settings:

TABLE 8-2 Mass Spectrometer Settings Test Article Q1/Q3 DP EP CE CXP ISTEM CAD CUR GS1 GS2 I-6 +562.6/282.4 133 10 29 19 5500 500 7 30 50 50I-2 +370.3/282.4 107 10 21 18 5500 500 7 30 50 50 I-3 +726.6/282.4 12910 38 18 5500 500 7 30 50 50 I-5 +584.5/282.4 135 10 24 20 5500 500 7 3050 50 I-11 +614.6/282.4 123 10 35 20 5500 500 7 30 50 50 I-7+724.5/282.4 120 10 40 19 5500 500 7 30 50 50 PEA +300.3/62.1  123 10 2910 5500 500 7 30 50 50

Column: Thermo BDS Hypersil C18 30 × 2.0 mm, 3 μm, with guard columnM.P. Buffer: 25 mM ammonium formate buffer,, pH 3.5 Aqueous Reservoir(A): 90% water, 10% buffer Organic Reservoir (B): 90% acetonitrile, 10%buffer Flow Rate: 350 μL/minute Gradient Program:

TABLE 8-3 Liquid Chromatography Gradient Program Time (mm) % A % B 0.0050 50 .080 25 75 1.50 0 100 2.00 0 100 2.10 50 50 3.00 50 50 Total RunTime: 3.0 minutes Autosampler: 10 μL injection volume Autosampler Wash:water/methanol/2-propanol: 1/1/1; with 0.2% formic acid MassSpectrometer Instrument: PE SCIEX API 4000 Interface: Turbo IonsprayMode: Multiple reaction monitoring Method: 3.0 minute duration Settings:

TABLE 8-4 Mass Spectrometer Settings Test Article Q1/Q3 DP EP CE CXP ISTEM CAD CUR GS1 GS2 I-9 +562.6/282.4 107 10 29 19 5500 500 7 20 20 30PEA +370.3/282.4 123 10 29 10 5500 500 7 20 20 30

Example 9: Determination of the Bioavailability of Palmitoylethanolamide(PEA) Following Oral Administration of PEA-Prodrug in MaleSprague-Dawley Rats

The present Example describes oral bioavailability of PEA followingadministration of PEA prodrugs in male Sprague-Dawley rats.

Oral bioavailability of palmitoylethanolamide (PEA) was evaluated inmale Sprague-Dawley rats following oral dosing of a PEA pro-drug, I-13.I-13 was dosed orally (PO) at 24.3 mg/kg, which is equivalent to a 10mg/kg dose of PEA. Blood samples were collected up to 8 hours post-dose,and plasma concentrations of PEA were determined by LC-MS/MS.Pharmacokinetic parameters, with the exception of C_(max) and t_(max)were not determined due to a lack of quantifiable data points. FollowingPO dosing of I-13 (in 20% (solutol HS15:NMP 1:1) 10% PEG400; 70% 120),nearly all rat plasma samples were below the limit of quantification.Maximum plasma concentrations (average of 2.70±0.0681 ng/mL) wereobserved between 2 and 8 hours post dosing. No ACUs or bioavailabilityvalues were determined.

Preparation for Dosing Formulations

Pro-drugs were dosed so that a total dose of 10 mg/kg of PEA wasadministered. Prodrugs were formulated in a vehicle comprised of 10%Solutol HS15, 10% n-methyl pyrrolidone (NMP), 10% polyethylene glycol400 (PEG400) and 70% water. Formulations were prepared fresh on the dayof dosing.

Animal Dosing

Pharmacokinetics of PEA were evaluated in fasted male Sprague-Dawleyrats. Rats were housed one per cage. Each rat was fitted with a jugularvein cannula (JVC) for blood collection. Each study group was dosing intriplicate. Rats were fasted for a minimum of twelve hours prior todosing. Food was returned at four hours post dosing. Animals had freeaccess to water throughout the study.

Blood samples (˜300 μL) were collected from the rats via a JVC andplaced into chilled polypropylene tubes containing sodium heparin as ananticoagulant, and 30 μL of 0.5 M citric acid. Samples were maintainedchilled throughout processing. Blood samples were centrifuged at 4° C.and 3,000 g for 5 minutes. Plasma (˜150 μL) was then transferred to achilled, labeled polypropylene tube containing 15 μL of 10% formic acid,placed on dry ice, and stored in a freezer maintained at −60° C. to −80°C. Blood sampling times are shown in Table 9a.

TABLE 9a Study Design. Dose Dosing Blood Total (mg/kg Solution DosingSample Test Dosing Animals of pro- Conc. Volume Time Article Route n =drug)* (mg/mL) (mL/kg) Vehicle Points I-13 PO 3 24.3 3 8.1 20% Pre-dose,(Solutol 5, 15, HS15:NMP 30 min, 1:1) 10% 1, 2, PEG400; 4, 8 hours 70%H₂O *All doses are based on mg/kg of the pro-drugs, and deliver 10 mg/kgof active drug, PEA.

An LC-MS/MS method for the determination of PEA and PEA-prodrug isdescribed above (see e.g., Example 3).

Pharmacokinetic parameters, with the exception of C_(max) and t_(max)were not determined due to a lack of quantifiable data points. Maximumplasma concentration (C_(max)) and time to reach maximum plasma drugconcentration (T_(max)) after oral dosing were observed from the data.Samples below the limit of quantitation were treated as zero forpharmacokinetic data analysis.

Results

No adverse reactions were observed following oral administration of PEApro-drug in male Sprague-Dawley rats in this study.

Dosing solutions were not analyzed by LC-MS/MS. Nominal dosing level wasused in all calculations. Individual and average plasma concentrationsfor PEA are shown in Table 9b. Data are expressed as ng/mL of the freedrug. Samples that were below the limit of quantitation were not used inthe calculation of averages. Plasma concentrations versus time data areplotted in FIG. 6. Endogenous levels of PEA found in all rats were belowlimit of quantitation; and therefore, measured concentrations of PEA inplasma samples were not corrected.

TABLE 9b Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-13 (in20% (Soluto HS15:NMP 1:1) 10% PEG400, 70% H₂O) at 24.3 mg/kg in MaleSprague-Dawley Rats. Oral (24.3 mg/kg I-13 equals 10 mg/kg PEA) Rat #Time (hr) 61 62 63 Mean SD 0(pre-dose) BLOQ BLOQ BLOQ ND ND 0.083 BLOQBLOQ BLOQ ND ND 0.25 BLOQ BLOQ BLOQ ND ND 0.50 BLOQ BLOQ BLOQ ND ND 1.0BLOQ BLOQ BLOQ ND ND 2.0 BLOQ 2.75 2.62 2.69 ND 4.0 BLOQ BLOQ BLOQ ND ND8.0 2.72 BLOQ BLOQ ND ND Animal Weight (kg)  0.239  0.241  0.228  0.2360.007 Volume Dosed (mL) 1.94 1.95 1.85 1.91 0.06 C_(max) (ng/mL) 2.722.75 2.62 2.70 0.0681 t_(max) (hr) 8.00 2.00 2.00 4.00 3.46 t_(1/2)(hr)ND¹ ND¹ ND¹ ND ND MRT_(last) (hr) ND¹ ND¹ ND¹ ND ND AUC_(last) (hrng/mL) ND¹ ND¹ ND¹ ND ND AUC_(∞) (hr ng/mL) ND¹ ND¹ ND¹ ND ND C_(max):maximum plasma concentration; t_(max): time of maximum plasmaconcentration; t_(1/2): half-life, data points used for half-lifedetermination are in bold; MRT_(last): mean residence time, calculatedto the last observable time point; AUC_(last): area under the curve,calculated to the last observable time point; AUC_(∞): area under thecurve, extrapolated to infinity; ND: not determined; BLOQ: below limitof quantitation (2.5 ng/mL); ¹not determined due to a lack ofquantifiable data points.

Example 10: PEA Stability in Human, Rat, Mouse and Dog Liver Microsomes,Human, Rat, Mouse and Dog Liver S9 Fraction, Human, Rat, Mouse and DogIntestinal S9 Fraction, Human, Rat, Mouse and Dog Plasma, and SimulatedIntestinal Fluid

The present Example describes PEA stability observed in 1) human, rat,mouse, and dog liver microsomes; 2) human, rat, mouse, dog liver S9fraction; human, rat, mouse, and dog intestinal S9 fraction; 4) human,rat, mouse, and dog plasma; and 5) simulated intestinal fluid containingvarious enzymes.

Liver Microsomal Stability

Mixed-gender human (Lot #1010420), male Sprague-Dawley rat (Lot#1510115), male CD-1 mouse (Lot #1510043), and male Beagle dog (Lot#0810143) liver microsomes were provided. Reaction mixture, minuscofactors, was prepared as described below. Test article was added intothe reaction mixture at a final concentration of 1 μM. Control compound,testosterone, were run simultaneously with the test article in aseparate reaction. An aliquot of the reaction mixture (without cofactor)was equilibrated in a shaking water bath at 37° C. for 5 minutes.Reaction was initiated by the addition of cofactor, and the mixture wasincubated in a shaking water bath at 37° C. Aliquots (100 μL) werewithdrawn at 0, 10, 20, 30, and 60 minutes. Test article samples wereimmediately combined with 300 μL of ice-cold acetonitrile containing 1%formic acid. Control samples were immediately combined with 400 μL ofice-cold 50/50 acetonitrile (ACN)/dH₂O containing 0.1% formic acid andinternal standard to terminate the reaction. Samples were then mixed andcentrifuged to precipitate proteins. Calibration standards were preparedin matched matrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both the dosed prodrug and the expected drug(PEA). Analytical conditions are outlined in Appendix 10-1. Test articleconcentration at each time point was compared to the test articleconcentration at time 0 to determine the percent remaining at each timepoint. Half-lives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 10aand 10b.

Reaction Composition

Liver Microsomes 0.5 mg/mL NADPH (cofactor) 1 mM Potassium Phosphate pH7.4 100 mM Magnesium Chloride 5 mM Test Article 1 μM

TABLE 10a PEA stability observed in human, rat, mouse, and dog livermicrosomes. CL_(int) ^(b) % Remaining of Initial (n = 1) Half- (mL/min/Test 10 20 30 60 life^(a) mg Article Species 0 min min min min min (min)protein) I-13 Human 100 79 73 60 42 47 0.0296 Rat 100 91 72 60 38 410.0336 Mouse 100 69 75 54 44 48 0.0291 Dog 100 78 76 56 32 38 0.0362^(a)When the calculated half-life is longer than the duration of theexperiment, half-life is expressed as > the longest incubation time.Similarly, if calculated half-life is less than the shortest time point,half-life is expressed as < that time point and calculated half-life isalso listed in parentheses. ^(b)Intrinsic clearance (CL_(int)) wascalculated based on CL_(int) = k/P, where k is elimination rate constantand P is protein concentration in the incubation.

Half- CL_(int) Acceptable Control life (ml/min/mg Range Compound Species(min) protein) (t_(1/2), min) Testosterone Human 21 0.0667 ≤41 Rat 1.41.01 ≤15 Mouse 7.3 0.190 ≤15 Dog 33 0.0419 ≤40

TABLE 10b Measured Concentrations of Drug. Dosed Concentration (μM) Test10 20 30 60 Article Species Analyte 0 min min min min min I-13 Human PEA0 0 0 0 0 Rat 0 0 0.013 0.013 0 Mouse 0 0 0 0 0 Dog 0 0 0 0 0

Liver S9 Stability

Mixed gender human (Lot #1210091), male Sprague-Dawley rat (Lot#1410265), male CD-1 mouse (Lot #1510255), and male Beagle dog (Lot#1310285) liver S9 fraction were provided. Reaction mixture, minuscofactors, was prepared as described below. Test article was added intothe reaction mixture at a final concentration of 1 μM. Controlcompounds, testosterone and 7-hydroxycoumarin (7-HC), were runsimultaneously with the test article in a separate reaction. An aliquotof the reaction mixture (without cofactor cocktail) was equilibrated ina shaking water bath at 37° C. for 5 minutes. Reaction was initiated bythe addition of cofactor cocktail (see below), and mixture was thenincubated in a shaking water bath at 37° C. Aliquots (100 tit) werewithdrawn at 0, 10, 20, 30, and 60 minutes. Test article samples wereimmediately combined with 300 μL of ice-cold acetonitrile containing 1%formic acid. Control samples were immediately combined with 400 μL ofice-cold 50/50 acetonitrile (ACN)/dH₂O containing 0.1% formic acid andinternal standard to terminate the reaction. Samples were then mixed andcentrifuged to precipitate proteins. Calibration standards were preparedin matched matrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both dosed prodrug and expected drug (PEA).Analytical conditions are outlined in Appendix 10-1. Test articleconcentration at each time point was compared to test articleconcentration at time 0 to determine the percent remaining at each timepoint. Half-lives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 10cand 10d.

Reaction Composition

Liver S9 Fraction 1.0 mg/mL NADPH (cofactor) 1 mM UDPGA (cofactor) 1 mMPAPS (cofactor) 1 mM GSH (cofactor) 1 mM Potassium Phosphate pH 7.4 100mM Magnesium Chloride 5 mM Test Article 1 μM

TABLE 10c PEA Stability observed in human, rat, mouse, and dog liver S9.CL_(int) ^(b) % Remaining of Initial (n = 1) Half- (mL/min/ Test 10 2030 60 life^(a) mg Article Species 0 min min min min min (min) protein)I-13 Human 100 93 87 78 45 58 0.0119 Rat 100 76 74 59 45 50 0.0138 Mouse100 89 90 79 78 >60 <0.0116 Dog 100 70 61 61 37 41 0.0169 ^(a)Whencalculated half-life is longer than the duration of the experiment,half-life is expressed as > the longest incubation time. Similarly, ifcalculated half-life is less than shortest time point, half-life isexpressed as < that time point and calculated half-life is also listedin parentheses. ^(b)Intrinsic clearance (CL_(int)) was calculated basedon CL_(int) = k/P, where k is the elimination rate constant and P is theprotein concentration in the incubation.

Half- CL_(int) Acceptable Control life (ml/min/mg Range Compound Species(min) protein) (t_(1/2), min) Testosterone Human 28 0.0247 ≤34 Rat 2.70.260 ≤15 Mouse 7.1 0.0976 ≤37 Dog 14 0.0493 ≤42 7-hydroxycourmarinHuman 15 0.0472 <18 Rat 1.9 0.362 <15 Mouse 3.8 0.182 <15 Dog 1.4 0.512<15

TABLE 10d Measured Concentrations of Drug. Dosed Concentration (μM) Test10 20 30 60 Article Species Analyte 0 min min min min min I-13 Human PEA0 0 0 0 0 Rat 0 0 0 0 0 Mouse 0 0 0 0 0 Dog 0.015 0.025 0.020 0.0240.021

Intestinal S9 Fraction Stability

Mixed-gender human (Lot #1410073), male Sprague-Dawley rat (Lot#1010042), male CD-1 mouse (Lot #1510194), and male Beagle dog (Lot#1510226) intestinal S9 fraction were provided. Reaction mixture, minuscofactors, was prepared as described below. Test article was added intothe reaction mixture at a final concentration of 1 μM. Controlcompounds, testosterone and 7-hydroxycoumarin, were run simultaneouslywith test article in a separate reaction. An aliquot of the reactionmixture (without cofactor cocktail) was equilibrated in a shaking waterbath at 37° C. for 5 minutes. Reaction was initiated by the addition ofcofactor cocktail, and mixture was incubated in a shaking water bath at37° C. Aliquots (100 μL) were withdrawn at 0, 10, 20, 30, and 60minutes. Test article samples were immediately combined with 300 μL ofice-cold acetonitrile containing 1% formic acid. Control samples wereimmediately combined with 400 μL of ice-cold 50/50 acetonitrile(ACN)/dH₂O containing 0.1% formic acid and internal standard toterminate the reaction. Samples were then mixed and centrifuged toprecipitate proteins. Calibration standards were prepared in matchedmatrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both dosed prodrug and expected drug (PEA).Analytical conditions are outlined in Appendix 10-1. Test articleconcentration at each time point was compared to test articleconcentration at time 0 to determine the percent remaining at each timepoint. Halflives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 10eand 10f.

Reaction Composition

Intestinal S9 Fraction 1.0 mg/mL NADPH (cofactor) 1 mM UDPGA (cofactor)1 mM PAPS (cofactor) 1 mM GSH (cofactor) 1 mM Potassium Phosphate, pH7.4 100 mM Magnesium Chloride 5 mM Test Article 1 μM

TABLE 10e PEA stability observed in human, rat, mouse, and dogintestinal S9 fraction. CL_(int) ^(b) % Remaining of Initial (n = 1)Half- (mL/min/ Test 10 20 30 60 life^(a) mg Article Species 0 min minmin min min (min) protein) I-13 Human 100 86 86 86 59 >60 (92) <0.0116(0.00757) Rat 100 80 82 81 55 >60 (81) <0.0116 (0.00859) Mouse 100 97 7181 57 >60 (73) <0.0116 (0.00951) Dog 100 67 63 46 49 45 0.0153 ^(a)Whencalculated half-life is longer than the duration of the experiment,half-life is expressed as > the longest incubation time. Similarly, ifcalculated half-life is less than shortest time point, half-life isexpressed as < that time point and calculated half-life is also listedin parentheses. ^(b)Intrinsic clearance (CL_(int)) was calculated basedon CL_(int) = k/P, where k is elimination rate constant and P is proteinconcentration in the incubation.

CL_(int) Control Half-life (ml/min/mg Compound Species (min) protein)Testosterone Human 14 0.0509 Rat >60 <0.0116 Mouse >60 <0.0116 Dog >60<0.0116 7-hydroxycourmarin Human 9.9 0.0698 Rat 22 0.0320 Mouse 4.30.160 Dog 8.7 0.0799

TABLE 10f Measured Concentrations of Drug. Dosed Concentration (μM) Test10 20 30 60 Article Species Analyte 0 min min min min min I-13 Human PEA0 0.010 0.014 0.018 0.031 Rat 0 0.020 0.048 0.064 0.092 Mouse 0.0120.050 0.073 0.096 0.089 Dog 0.058 0.095 0.10 0.089 0.17

Plasma Stability

Studies were carried out in mixed-gender human plasma (Lot #AS1650-2),male Sprague-Dawley rat (Lot #RAT297944), male CD-I mouse (Lot#MSE237700), and male Beagle dog (Lot #BGL91384), collected on sodiumheparin. Plasma was adjusted to pH 7.4 prior to initiating theexperiments. DMSO stocks were first prepared for the test articles.Aliquots of the DMSO solutions were dosed into 700 μL of plasma, whichhad been pre-warmed to 37° C., at a final test article concentration of1 μM. Aliquots (100 μL) were taken at each time point (0, 15, 30, 60,and 120 minutes) and were immediately combined with 300 μL of ice-coldacetonitrile containing 1% formic acid. Samples were stored at 4° C.until the end of the experiment. After the final time point was sampled,the plate was mixed and then centrifuged at 3,000 rpm for 10 minutes.Calibration standards were prepared in matched matrix. Samples andstandards were assayed by LC-MS/MS using electrospray ionization forboth dosed prodrug and expected drug (PEA). Analytical conditions areoutlined in Appendix 10-1. Test article concentration at each time pointwas compared to test article concentration at time 0 to determine thepercent remaining at each time point. Half-lives were calculated usingGraphPad software, fitting to a single-phase exponential decay equation.Results are shown in Tables 10g and 10h.

TABLE 10g PEA stability observed in human, rat, mouse, and dog plasma. %Remaining of Initial (n = 1) Half- Test 15 30 60 120 life^(a) ArticleSpecies 0 min min min min min (min) I-13 Human 100 104 115 95 40 120 Rat100 69 76 36 5.1 42 Mouse 100 82 71 49 35 70 Dog 100 90 72 54 23 61

TABLE 10h Measured Concentrations of Drug. Dosed Concentration (μM) Test15 30 60 120 Article Species Analyte 0 min min min min min I-13 HumanPEA 0 0 0 0 0 Rat 0 0 0 0.022 0.053 Mouse 0 0.016 0.037 0.068 0.090 Dog0 0 0 0 0

Simulated Intestinal Fluid Stability

Studies were carried out in simulated intestinal fluid in the presenceof various enzymes. Simulated intestinal fluid was prepared bydissolving 6.8 g of monobasic potassium phosphate in 1.0 L of water.Aliquots of this solution were taken and the pH was adjusted to 6.8.Individual enzymes were then spiked into aliquots for each experiment. ADMSO stock was first prepared for the test article. Aliquots of the DMSOsolution were dosed into 700 μL of matrix, which had been pre-warmed to37° C., at a final test article concentration of 1 μM. Aliquots (100 μL)were taken at each time point (0, 15, 30, 60, and 120 minutes) and wereimmediately combined with 300 μL of ice-cold acetonitrile containing 1%formic acid. Samples were stored at 4° C. until the end of theexperiment. After the final time point was sampled, the plate was mixedand then centrifuged at 3,000 rpm for 10 minutes. Calibration standardswere prepared in matched matrix. Samples and standards were assayed byLC-MS/MS using electrospray ionization for both dosed prodrug andexpected drug (PEA). Analytical conditions are outlined in Appendix10-1. Test article concentration at each time point was compared to testarticle concentration at time 0 to determine the percent remaining ateach time point. Half-lives were calculated using GraphPad software,fitting to a single-phase exponential decay equation. Results are shownin Tables 10i and 10j.

TABLE 10i PEA Stability observed in simulated intestinal fluid (SIF). %Remaining of Initial (n = 1) Half- Test 15 30 60 120 life^(a) ArticleTreatment 0 min min min min min (min) I-13 SIF + Pancreatin 100 1.3 3.73.4 <1.0 <15 (2.5) SIF + Elastase 100 41 16 5.6 5.0 12 SIF +Carboxypeptidase A 100 77 53 27 37 47 SIF + Carboxypeptidase B 100 65 3114 8.7 20 SIF + Chymotrypsin 100 82 67 45 15 49 SIF + Trypsin 100 78 7055 <1.0 47

TABLE 10j Measured Concentrations of Drug. Concentration (μM) Test 15 3060 120 Article Treatment Analyte 0 min min min min min I-13 SIF +Pancreatin PEA — — — — — SIF + Elastase 0 0 0 0 0 SIF + CarboxypeptidaseA 0 0 0 0 0 SIF + Carboxypeptidase B 0 0 0 0 0 SIF + Chymotrypsin 0 0 00 0 SIF + Trypsin 0 0 0 0 0

PEA was found to be endogenous in pancreatin and thus could not bequantified in the assay samples.

Appendix 10-1 Liquid Chromatography

Column: Waters ACQUITY UPLC BEH C18 30 × 2.1 mm, 1. M.P. Buffer: 25 mMammonium formate buffer,, pH 3.5 Aqueous Reservoir (A): 90% water, 10%buffer Organic Reservoir (B): 90% acetonitrile, 10% buffer Flow Rate:0.8 mL/minute Gradient Program:

TABLE 10-1 Liquid Chromatography Gradient Program Time (min) % A % B0.00 50 50 0.75 1 99 1.50 1 99 1.55 50 50 2.00 50 50 Total Run Time: 2.0minutes Autosampler: 10 μL injection volume Wash1:water/methanol/2-propanol: 1/1/1; with 0.2% formic acid Wash2: 0.1%formic acid in water Mass Spectrometer Instrument: PE SCIEX API 4000Interface: Turbo Ionspray Mode: Multiple reaction monitoring Method: 2.0minute duration Settings:

Table 10-2: Mass Spectrometer Settings

TABLE 10-2 Mass Spectrometer Settings Test Article Q1/Q3 DP EP CE CXP ISTEM CAD CUR GS1 GS2 I-13  +998.9/282.2 141 10 68 28 5500 500 7 30 50 50+1015.9/282.2 42 10 53 7 5500 500 7 30 50 50 +1015.9/998.9 63 10 20 175500 500 7 30 50 50 PEA +300.3/62.1 123 10 29 10 5500 500 7 30 50 50

Example 11: PEA Stability in Simulated Intestinal Fluid SimulatedIntestinal Fluid Stability

Studies were carried out in simulated intestinal fluid containingpancreatin. Simulated intestinal fluid was prepared by dissolving 6.8 gof monobasic potassium phosphate in 1.0 L of water. Pancreatin was thenadded to the solution and the pH was adjusted to 6.8. A DMSO stock wasfirst prepared for the test articles. Aliquots of the DMSO solution weredosed into 300 μL of matrix, which had been pre-warmed to 37° C., at afinal test article concentration of 1 μM. An individual tube was dosedfor each time point. At each time point (0, 15, 30, 60, and 120minutes), 900 μL of ice-cold acetonitrile containing 1.0% formic acidwas added to an individual tube. Starting time for each tube wasstaggered such that all timepoints would finish at the same time. Afterthe conclusion of the experiment, tubes were mixed and then centrifugedat 3,000 rpm for 10 minutes. Calibration standards were prepared inmatched matrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization. Analytical conditions are outlined in Appendix11-1. Test article concentration at each time point was compared to testarticle concentration at time 0 to determine the percent remaining ateach time point. Halflives were calculated using GraphPad software,fitting to a single-phase exponential decay equation. Results are shownin Tables 11a and 11b.

TABLE 11a PEA stability observed in simulated intestinal fluid (SIF) %Remaining of Initial (n = 1) Half- Test 15 30 60 120 life^(a) ArticleTreatment 0 min min min min min (min) I-15 SIF + Pancreatin 100 <2.2<2.2 <2.2 <2.2 ND I-14 SIF + Pancreatin 100 25 <2.2 <2.2 <2.2 7.6

TABLE 11b Measured Concentrations of Drug. Concentration (μM) Test 15 3060 120 Article Treatment 0 min min min min min I-15 SIF + Pancreatin0.18 0 0 0 0 I-14 SIF + Pancreatin 0.19 0.047 0 0 0

Appendix 11-1 Liquid Chromatography

Column: Waters ACQUITY UPLC BEH C18 × 2.1 mm, 1.7 μm M.P. Buffer: 25 mMammonium formate buffer,, pH 3.5 Aqueous Reservoir (A): 90% water, 10%buffer Organic Reservoir (B): 90% acetonitrile, 10% buffer Flow Rate:0.7 mL/minute Gradient Program:

TABLE 11-1 Liquid Chromatography Gradient Program Time (min) % A % B0.00 50 50 0.75 1 99 1.00 1 99 1.05 50 50 1.50 50 50 Total Run Time: 1.5minutes Autosampler: 1 μL injection volume Wash1:water/methanol/2-propanol: 1/1/1; with 0.2% formic acid Wash2: 0.1%formic acid in water Mass Spectrometer Instrument: PE SCIEX API 4000Interface: Turbo Ionspray Mode: Multiple reaction monitoring Method: 1.5minute duration Settings:

TABLE 11-2 Mass Spectrometer Settings Test Article Q1/Q3 DP EP CE CXP ISTEM CAD CUR GS1 GS1 I-15 +614.5/282.6 112 10 40 7 5500 500 7 30 50 50I-14 +614.5/282.8 124 10 30 8 5500 500 7 30 50 50

Example 12: Determination of the Bioavailability ofPalmitoylethanolamide (PEA) Following Oral Administration of PEA-Prodrugin Male Sprague-Dawley Rats

The present Example describes oral bioavailability of PEA followingadministration of PEA prodrugs in male Sprague-Dawley rats.

Oral bioavailability of palmitoylethanolamide (PEA) was evaluated inmale Sprague-Dawley rats following oral dosing of a PEA pro-drug, I-12.I-12 was dosed orally (PO) at 35.2 mg/kg in two different formulations,which is equivalent to a 10 mg/kg dose of PEA. Blood samples werecollected up to 8 hours post-dose, and plasma concentrations of PEA weredetermined by LC-MS/MS. Following PO dosing of Group 1 of I-12 (in 20%(Solutol HS15:NMP 1:1) 10% PEG400; 70% H₂O) with analysis of PEA,maximum plasma concentrations (average of 12.8±1.68 ng/mL) were observedat 1 hour post dosing. Average half-life could not be determined due toa lack of quantifiable data points trailing the Cma*. Average exposurefor PEA based on the dose-normalized AUC_(last) was 2.23±1.08hr*kg*ng/mL/mg. Based on the IV data from Example 5, average oralbioavailability for PEA (Group 1) was 3.60±1.73%. Following PO dosing ofGroup 2 of I-12 (in 0.5% methyl cellulose in 20% (Solutol HS15:NMP 1:1);10% PEG400: 70% H20) with analysis of PEA, maximum plasma concentrations(average of 16.1±3.62 ng/mL) were observed at 1 hour post dosing.Average half-life after PO dosing could not be determined; however,half-life for one rat was 4.34 hours. Average exposure for PEA based onthe dose-normalized AUC_(last) was 3.43±1.03 hr*kg*ng/mL/mg. Based onthe IV data from Example 5, average oral bioavailability for PEA (Group2) was 5.52±1.66%.

Preparation of Dosing Formulations

Pro-drugs were dosed so that a total dose of 10 mg/kg of PEA wasadministered. Pro-drugs were formulated in a vehicle comprised of 10%Solutol HS15, 10% n-methyl pyrrolidone (NMP), 10% polyethylene glycol400 (PEG400) and 70% water for Group 1 or in in a vehicle comprised of0.5% methyl cellulose in 10% Solutol HS15, 10% NMP, 10% PEG400 and 70%water (Group 2). Formulations were prepared fresh on the day of dosing.

Animal Dosing

Pharmacokinetics of PEA were evaluated in fasted male Sprague-Dawleyrats. Rats were housed one per cage. Each rat was fitted with a jugularvein cannula (JVC) for blood collection. Each study group was dosing intriplicate. Rats were fasted for a minimum of twelve hours prior todosing. Food was returned at four hours post dosing. Animals had freeaccess to water throughout the study. Blood samples (˜300 μL) werecollected from rats via a JVC and placed into chilled polypropylenetubes containing sodium heparin as an anticoagulant, and 30 μL of 0.5 Mcitric acid. Samples were maintained chilled throughout processing.Blood samples were centrifuged at 4° C. and 3,000 g for 5 minutes.Plasma (˜150 μL) was then transferred to a chilled, labeledpolypropylene tube containing 15 μL of 10% formic acid, placed on dryice, and stored in a freezer maintained at −60° C. to −80° C. Bloodsampling times are shown in Table 12a.

TABLE 12a Study Design. Dose Dosing Blood Total (mg/kg Solution DosingSample Dose Test Dosing Animals of pro- Conc. Volume Time group ArticleRoute n = drug)* (mg/mL) (mL/kg) Vehicle Points 1 I-12 PO 3 35.2 5.03 720% Pre-dose, (Solutol 5, 15, HS15:NMP 30 min, 1:1) 10% 1, 2, PEG400; 4,8 hours 70% H₂O 2 PO 3 35.2 5.03 7 20% Pre-dose, (Solutol 5, 15,HS15:NMP 30 min, 1:1) 10% 1, 2, PEG400; 4, 8 hours 70% H₂O *All dosesare based on mg/kg of the pro-drugs, and deliver 10 mg/kg of activedrug, PEA.

An LC-MS/MS method for the determination of PEA and PEA-prodrug isdescribed above (see e.g., Example 3).

Pharmacokinetic parameters were calculated from the time course of theplasma concentration. Maximum plasma concentration (C_(max)) and time toreach maximum plasma drug concentration (T_(max)) after oral dosing wereobserved from the data. Area under the time concentration curve (AUC)was calculated using the linear trapezoidal rule with calculation to thelast quantifiable data point, and with extrapolation to infinity ifapplicable. At least three quantifiable data points were required todetermine the AUC. Plasma half-life (t_(1/2)) was calculated from0.693/slope of the terminal elimination phase. Mean residence time, MRT,was calculated by dividing area under the moment curve (AUMC) by theAUC. Bioavailability was determined by dividing the individualdose-normalized PO AUC_(last) values by the average IV AUC_(last) value(IV data from Example 5). Samples below the limit of quantitation weretreated as zero for pharmacokinetic data analysis.

Results

No adverse reactions were observed following the oral administration ofPEA pro-drug in male Sprague-Dawley rats in this study.

Dosing solutions were not analyzed by LC-MS/MS. Nominal dosing level wasused in all calculations. Individual and average plasma concentrationsfor PEA and are shown in Tables 12b and 12c. Data are expressed as ng/mLof the free drug. Samples that were below the limit of quantitation werenot used in the calculation of averages. Plasma concentrations versustime data are plotted in FIGS. 7A through 7D. Endogenous levels of PEAfound in all rats were below the limit of quantitation; and therefore,measured concentrations of PEA in plasma samples were not corrected.

TABLE 12b Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-12 (in20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 35.2 mg/kg in MaleSprague-Dawley Rats (Group 1) Oral (35.2 mg/kg I-12 equals 10 mg/kg PEA)Rat # Time (hr) 13 14 15 Mean SD 0 (pre-dose) BLOQ BLOQ BLOQ ND ND 0.083BLOQ BLOQ BLOQ ND ND 0.25 3.84 5.64 3.69 4.39 1.09 0.50 7.08 10.2  9.068.78 1.58 1.0 11.9  11.7  14.7  12.8  1.68 2.0 6.14 6.11 10.1  7.45 2.304.0 BLOQ BLOQ 4.40 ND ND 8.0 BLOQ BLOQ BLOQ ND ND Animal Weight (kg) 0.291  0.279  0.277  0.282  0.008 Volume Dosed (mL) 2.04 1.95 1.94 1.980.06 C_(max) (ng/mL) 11.9  11.7  14.7  12.8  1.68 t_(max) (hr) 1.00 1.001.00 1.00 0.00 t_(1/2)(hr) ND³ ND³ ND³ ND ND MRT_(last) (hr) 1.07 1.021.75 1.28  0.409 AUC_(last) (hr ng/mL) 15.5  16.8  34.7  22.3  10.8 AUC_(∞) (hr ng/mL) ND³ ND³ ND³ ND ND Dose-normalized Values¹ AUC_(last)(hr kg 1.55 1.68 3.47 2.23 1.08 ng/mL/mg) AUC_(∞) (hr kg ng/mL/mg) ND³ND³ ND³ ND ND Bioavailability (%)² 2.49 2.71 5.60 3.60 1.73 C_(max):maximum plasma concentration; t_(max): time of maximum plasmaconcentration; t_(1/2): half-life, data points used for half-lifedetermination are in bold; MRT_(last): mean residence time, calculatedto the last observable time point; AUC_(last): area under the curve,calculated to the observable time point; AUC_(∞): area under the curve,extrapolated to infinity; ND: not determined; BLOQ: below the limit ofquantitation (2.5 ng/mL); ¹Dosenormalized by dividing the parameter bythe nominal dose in mg/kg; ²Bioavailability determined by dividing theindividual dose-normalized oral AUC_(last) values by the average IVAUC_(last) value 62.1 hr*ng/mL from Example 5; ³not determined due tolack of quantifiable data points trailing the C_(max).

TABLE 12c Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-12 (in0.5% Methyl Cellulose in 20% (Solutol HS15:NMP (1:1), 10% PEG400, 70%H₂O) at 35.2 mg/kg in Male Sprague-Dawley Rats (Group 2). Oral (35.2mg/kg I-12 equals 10 mg/kg PEA) Rat # Time (hr) 16 17 18 Mean SD 0(pre-dose) BLOQ BLOQ BLOQ ND ND 0.083 BLOQ BLOQ BLOQ ND ND 0.25 5.793.55 2.98 4.11 1.49 0.50 8.27 9.21 7.72 8.40  0.753 1.0 20.1  13.0 15.3  16.1  3.62 2.0 6.78 2.71 6.56 5.35 2.29 4.0 3.96 BLOQ 5.02 4.49 ND8.0 BLOQ 3.17 2.54 2.86 ND Animal Weight (kg)  0.283  0.282  0.275 0.280  0.004 Volume Dosed (mL) 1.98 1.97 1.93 1.96 0.03 C_(max) (ng/mL)20.1  13.0  15.3  16.1  3.62 t_(max) (hr) 1.00 1.00 1.00 1.00 0.00t_(1/2)(hr) ND³ ND³ 4.34 ND ND MRT_(last) (hr) 1.58 2.90 2.97 2.48 0.780 AUC_(last) (hr ng/mL) 33.5  24.3  45.0  34.3  10.3  AUC_(∞) (hrng/mL) ND³ ND³ ND⁴ ND ND Dose-normalized Values¹ AUC_(last) (hr kg 3.352.43 4.50 3.43 1.03 ng/mL/mg) AUC_(∞) (hr kg ng/mL/mg) ND³ ND³ ND⁴ ND NDBioavailability (%)² 5.40 3.92 7.24 5.52 1.66 C_(max): maximum plasmaconcentration; t_(max): time of maximum plasma concentration; t_(1/2):half- life, data points used for half-life determination are in bold;MRT_(last): mean residence time, calculated to the last observable timepoint; AUC_(last): area under the curve, calculated to the lastobservable time point; AUC_(∞): area under the curve, extrapolated toinfinity; ND: not determined; BLOQ: below the limit of quantitation (2.5ng/mL); ¹Dose-normalized by dividing the parameter by nominal dose inmg/kg; ²Bioavailability determined by dividing individualdose-normalized oral AUC_(last) values by the average IV AUC_(last)value 62.1 hr*ng/mL from Example 5; ³not determined due to lack ofquantifiable data points trailing the C_(max); ⁴not determined becausethe AUC_(∞) was a greater than 25% extrapolation above the AUC_(last).

Example 13: PEA Stability in Human, Rat Mouse and Dog Liver Microsomes,Human, Rat, Mouse and Dog Liver S9 Fraction, Human, Rat, Mouse and DogIntestinal S9 Fraction, Human, Rat, Mouse and Dog Plasma, and SimulatedIntestinal Fluid

The present Example describes PEA stability observed in 1) human, rat,mouse, and dog liver microsomes; 2) human, rat, mouse, dog liver S9fraction; human, rat, mouse, and dog intestinal S9 fraction; 4) human,rat, mouse, and dog plasma; and 5) simulated intestinal fluid containingvarious enzymes.

Liver Microsomal Stability

Mixed-gender human (Lot #1010420), male Sprague-Dawley rat (Lot#1510115), male CD-1 mouse (Lot #1610148), and male Beagle dog (Lot#0810143) liver microsomes were provided. Reaction mixture, minuscofactors, was prepared as described below. Test article was added intothe reaction mixture at a final concentration of 1 μM. Control compound,testosterone, was run simultaneously with the test article in a separatereaction. An aliquot of the reaction mixture (without cofactor) wasequilibrated in a shaking water bath at 37° C. for 3 minutes. Reactionwas initiated by the addition of cofactor, and the mixture was incubatedin a shaking water bath at 37° C. Aliquots (100 μL) were withdrawn at 0,10, 20, 30, and 60 minutes. Test article samples were immediatelycombined with 300 μL, of ice-cold acetonitrile containing 1% formicacid. Control samples were immediately combined with 400 μL of ice-cold50/50 acetonitrile (ACN)/dH₂O containing 0.1% formic acid and internalstandard to terminate the reaction. Samples were then mixed andcentrifuged to precipitate proteins. Calibration standards were preparedin matched matrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both dosed prodrug and expected drug (PEA).Analytical conditions are outlined in Appendix 13-1. Test articleconcentration at each time point was compared to test articleconcentration at time 0 to determine the percent remaining at each timepoint. Half-lives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 13aand 13b.

Reaction Composition

Liver Microsomes 0.5 mg/mL NADPH (cofactor) 1 mM Potassium Phosphate pH7.4 100 mM Magnesium Chloride 5 mM Test Article 1 μL

TABLE 13a PEA stability observed in human, rat, mouse, and dog livermicrosomes. CL_(int) ^(b) % Remaining of Initial (n = 1) Half- (mL/min/Test 10 20 30 60 life^(a) mg Article Species 0 min min min min min (min)protein) I-12 Human 100 105 96 91 73 >60 <0.0231 Rat 100 85 77 62 52 580.0240 Mouse 100 83 88 68 61 >60 (82) <0.0231 (0.0169) Dog 100 93 79 7860 >60 (80) <0.0231 (0.0173) ^(a)When the calculated half-life is longerthan the duration of the experiment, half-life is expressed as > thelongest incubation time. Similarly, if calculated half-life is less thanthe shortest time point, half-life is expressed as < that time point andcalculated half-life is also listed in parentheses. ^(b)Intrinsicclearance (CL_(int)) was calculated based on CL_(int) = k/P, where k iselimination rate constant and P is protein concentration in theincubation.

Half- CL_(int) Acceptable Control life (ml/min/mg Range Compound Species(min) protein) (t_(1/2), min) Testosterone Human 21 0.0667 ≤41 Rat 1.41.01 ≤15 Mouse 3.1 0.444 ≤15 Dog 33 0.0419 ≤40

TABLE 13b Measured Concentrations of Drug. Dosed Concentration (μM) Test10 20 30 60 Article Species Analyte 0 min min min min min I-12 Human PEA0 0 0 0 0 Rat 0.022 0.022 0.015 0.011 0.0048 Mouse 0.037 0.038 0.0300.022 0.011 Dog 0.019 0.032 0.036 0.035 0.033

Liver S9 Stability

Mixed gender human (Lot #1210091), male Sprague-Dawley rat (Lot#1410265), male CD-1 mouse (Lot #1510255), and male Beagle dog (Lott#1310285) liver S9 fraction were provided. The reaction mixture, minuscofactors, was prepared as described below. Test article was added intothe reaction mixture at a final concentration of 1 μM. Controlcompounds, testosterone and 7-hydroxycoumarin (7-HC), were runsimultaneously with the test article in a separate reaction. An aliquotof the reaction mixture (without cofactor cocktail) was equilibrated ina shaking water bath at 37° C. for 3 minutes. Reaction was initiated bythe addition of cofactor cocktail (see below), and the mixture was thenincubated in a shaking water bath at 37° C. Aliquots (100 μL) werewithdrawn at 0, 10, 20, 30, and 60 minutes. Test article samples wereimmediately combined with 300 μL of ice-cold acetonitrile containing 1%formic acid. Control samples were immediately combined with 400 μL ofice-cold 50/50 acetonitrile (ACN)/dH₂O containing 0.1% formic acid andinternal standard to terminate the reaction. Samples were then mixed andcentrifuged to precipitate proteins. Calibration standards were preparedin matched matrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both dosed prodrug and expected drug (PEA).Analytical conditions are outlined in Appendix 13-1. Test articleconcentration at each time point was compared to the test articleconcentration at time 0 to determine the percent remaining at each timepoint. Half-lives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 13cand 13d.

Reaction Composition

Liver S9 Fraction 1.0 mg/mL NADPH (cofactor) 1 mM UDPGA (cofactor) 1 mMPAPS (cofactor) 1 mM GSH (cofactor) 1 mM Potassium Phosphate pH 7.4 100mM Magnesium Chloride 5 mM Test Article 1 μM

TABLE 13c PEA stability observed in human, rat, mouse, and dog liver S9.CL_(int) ^(b) % Remaining of Initial (n = 1) Half- (mL/min/ Test 10 2030 60 life^(a) mg Article Species 0 min min min min min (min) protein)I-12 Human 100 89 73 68 43 49 0.0141 Rat 100 82 85 75 61 >60 (90)<0.0116 (0.00769) Mouse 100 75 66 58 50 53 0.0130 Dog 100 69 53 51 47 430.0161 ^(a)When calculated half-life is longer than the duration of theexperiment, half-life is expressed as > the longest incubation time.Similarly, if calculated half-life is less than shortest time point,half-life is expressed as < that time point and calculated half-life isalso listed in parentheses. ^(b)Intrinsic clearance (CL_(int)) wascalculated based on CL_(int) = k/P, where k is the elimination rateconstant and P is the protein concentration in the incubation.

Half- CL_(int) Acceptable Control life (ml/min/mg Range Compound Species(min) protein) (t_(1/2); min) Testosterone Human 28 0.0247 ≤34 Rat 2.70.260 ≤15 Mouse 9.0 0.0770 ≤37 Dog 14 0.0493 ≤42 7-hydroxycourmann Human15 0.0472 <18 Rat 1.9 0.362 <15 Mouse 2.2 0.313 <15 Dog 1.4 0.512 <15

TABLE 13d Measured Concentrations of Drug. Dosed Concentration (μM) Test10 20 30 60 Article Species Analyte 0 min min min min min I-12 Human PEA0.016 0.042 0.047 0.040 0.035 Rat 0 0.018 0.017 0.020 0 Mouse 0.0370.049 0.041 0.033 0.021 Dog 0.026 0.047 0.053 0.051 0.033

Intestinal S9 Fraction Stability

Mixed-gender human (Lot #1410073), male Sprague-Dawley rat (Lot#1010042), male CD-1 mouse (Lot #1510194), and male Beagle dog (Lot#1510226) intestinal S9 fraction were provided. Reaction mixture, minuscofactors, was prepared as described below. Test article was added intothe reaction mixture at a final concentration of 1 μM. Controlcompounds, testosterone and 7-hydroxycoumarin, were run simultaneouslywith the test article in a separate reaction. An aliquot of the reactionmixture (without cofactor cocktail) was equilibrated in a shaking waterbath at 37° C. for 3 minutes. Reaction was initiated by the addition ofcofactor cocktail, and the mixture was incubated in a shaking water bathat 37° C. Aliquots (100 μL) were withdrawn at 0, 10, 20, 30, and 60minutes. Test article samples were immediately combined with 300 μL ofice-cold acetonitrile containing 1% formic acid. Control samples wereimmediately combined with 400 μL of ice-cold 50/50 acetonitrile(ACN)/dH₂O containing 0.1% formic acid and internal standard toterminate the reaction. Samples were then mixed and centrifuged toprecipitate proteins. Calibration standards were prepared in matchedmatrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both dosed prodrug and expected drug (PEA).Analytical conditions are outlined in Appendix 13-1. Test articleconcentration at each time point was compared to test articleconcentration at time 0 to determine the percent remaining at each timepoint. Halflives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 13eand 13f.

Reaction Composition

Intestinal S9 Fraction 1.0 mg/mL NADPH (cofactor) 1 mM UDPGA (cofactor)1 mM PAPS (cofactor) 1 mM GSH (cofactor) 1 mM Potassium Phosphate, pH7.4 100 mM Magnesium Chloride 5 mM Test Article 1 μM

TABLE 13e PEA stability observed in human, rat, mouse, and dogintestinal S9 fraction. CL_(int) ^(b) % Remaining of Initial (n = 1)Half- (mL/min/ Test 10 20 30 60 life^(a) mg Article Species 0 min minmin min min (min) protein) I-12 Human 100 98 82 79 65 >60 (89) <0.0116(0.00782) Rat 100 92 88 74 53 >60 (67) <0.0116 (0.0103)  Mouse 100 80 5864 43 46 0.0151 Dog 100 61 44 48 47 41 0.0169 ^(a)When calculatedhalf-life is longer than the duration of the experiment, half-life isexpressed as > the longest incubation time. Similarly, if calculatedhalf-life is less than shortest time point, half-life is expressed as <that time point and calculated half-life is also listed in parentheses.^(b)Intrinsic clearance (CL_(int)) was calculated based on CL_(int) =k/P, where k is elimination rate constant and P is protein concentrationin the incubation.

CL_(int) Control Half-life (ml/min/mg Compound Species (min) protein)Testosterone Human 14 0.0509 Rat >60 <0.0116 Mouse >60 <0.0116 Dog >60<0.0116 7-hydroxycourmarin Human 9.9 0.0698 Rat 22 0.0320 Mouse 4.30.160 Dog 8.7 0.0799

TABLE 13f Measured Concentrations of Drug. Dosed Concentration (μM) Test10 20 30 60 Article Species Analyte 0 min min min min min I-12 Human PEA0.0089 0.023 0.024 0.032 0.043 Rat 0 0.039 0.056 0.074 0.10 Mouse 0.0220.084 0.11 0.15 0.16 Dog 0.088 0.19 0.22 0.23 0.26

Plasma Stability

Studies were carried out in mixed-gender human plasma (Lot #AS1650-2),male Sprague-Dawley rat (Lot #RAT297944), male CD-1 mouse (Lot#MSE237700), and male Beagle dog (Lot #BGL91384), collected on sodiumheparin. Plasma was adjusted to pH 7.4 prior to initiating theexperiments. DMSO stocks were first prepared for test articles. Aliquotsof the DMSO solutions were dosed into 700 μL of plasma, which had beenpre-warmed to 37° C., at a final test article concentration of 1 μM,Aliquots (100 μL) were taken at each time point (0, 15, 30, 60, and 120minutes) and were immediately combined with 300 μL of ice-coldacetonitrile containing 1% formic acid. Samples were stored at 4° C.until the end of experiment. After the final time point was sampled, theplate was mixed and then centrifuged at 3,000 rpm for 10 minutes.Calibration standards were prepared in matched matrix. Samples andstandards were assayed by LC-MS/MS using electrospray ionization forboth dosed prodrug and the expected drug (PEA). Analytical conditionsare outlined in Appendix 13-1. Test article concentration at each timepoint was compared to test article concentration at time 0 to determinethe percent remaining at each time point. Half-lives were calculatedusing GraphPad software, fitting to a single-phase exponential decayequation. Results are shown in Tables 13g and 13h.

TABLE 13g PEA stability observed in human, rat mouse, and dog plasma. %Remaining of Initial (n = 1) Half- Test 15 30 60 120 life^(a) ArticleSpecies 0 min min min min min (min) I-12 Human 100 114 123 87 55 >120(137) Rat 100 85 71 89 61 >120 (216) Mouse 100 104 107 74 42 102 Dog 10085 99 88 48 >120 (146)

TABLE 13h Measured Concentrations of Drug. Dosed Concentration (μM) Test15 30 60 120 Article Species Analyte 0 min min min min min I-12 HumanPEA 0 0.016 0.026 0.025 0.042 Rat 0.031 0.059 0.073 0.099 0.13 Mouse0.046 0.073 0.094 0.098 0.10 Dog 0 0.023 0.027 0.039 0.048

Simulated Intestinal Fluid Stability

Studies were carried out in simulated intestinal fluid in the presenceof various enzymes. Simulated intestinal fluid was prepared bydissolving 6.8 g of monobasic potassium phosphate in 1.0 L of water.Aliquots of this solution were taken and the pH was adjusted to 6.8.Individual enzymes were then spiked into aliquots for each experiment. ADMSO stock was first prepared for test article. Aliquots of the DMSOsolution were dosed into 700 μL of matrix, which had been pre-warmed to37° C., at a final test article concentration of 1 μM. Aliquots (100 μL)were taken at each time point (0, 15, 30, 60, and 120 minutes) and wereimmediately combined with 300 μL of ice-cold acetonitrile containing 1%formic acid. Samples were stored at 4° C. until the end of experiment.After final time point was sampled, the plate was mixed and thencentrifuged at 3,000 rpm for 10 minutes. Calibration standards wereprepared in matched matrix. Samples and standards were assayed byLC-MS/MS using electrospray ionization for both dosed prodrug andexpected drug (PEA). Analytical conditions are outlined in Appendix13-1. Test article concentration at each time point was compared to testarticle concentration at time 0 to determine the percent remaining ateach time point. Half-lives were calculated using GraphPad software,fitting to a single-phase exponential decay equation. Results were shownin Tables 13i and 13j.

TABLE 13i PEA stability observed in simulated intestinal fluid (SIF). %Remaining of Initial (n = 1) Half- Test 15 30 60 120 life^(a) ArticleTreatment 0 min min min min min (min) I-12 SIF + Pancreatin 100 24 144.7 <1.0 <15 (8.2) SIF + Elastase 100 101 88 63 33 79 SIF +Carboxypeptidase A 100 74 71 25 12 38 SIF + Carboxypeptidase B 100 71 4824 3.4 28 SIF + Chymotrypsin 100 86 64 31 4.1 37 SIF + Trypsin 100 77 5564 37 86

TABLE 13j Measured Concentrations of Drug. Concentration (μM) Test 15 3060 120 Article Treatment Analyte 0 min min min min min I-12 SIF +Pancreatin PEA — — — — — SIF + Elastase 0 0 0 0 0 SIF + CarboxypeptidaseA 0 0 0 0 0 SIF + Carboxypeptidase B 0 0 0 0 0 SIF + Chymotrypsin 0 0 00 0 SIF + Trypsin 0 0 0 0 0

PEA was found to be endogenous in pancreatin and thus could not bequantified in the assay samples.

Appendix 13-1 Liquid Chromatography

Column: Waters ACQUITY UPLC BEH C18 30 × 2.1 mm, 1.7 μm M.P. Buffer: 25mM ammonium formate buffer,, pH 3.5 Aqueous Reservoir (A): 90% water,10% buffer Organic Reservoir (B): 90% acetonitrile, 10% buffer FlowRate: 0.8 mL/minute Gradient Program:

TABLE 13-1 Liquid Chromatography Gradient Program Time (mm) % A % B 0.0050 50 0.75 1 99 1.50 1 99 1.55 50 50 1.75 50 50 Total Run Time: 1.75minutes Autosampler: 10 μL injection volume Wash1:water/methanol/2-propanol: 1/1/1; with 0.2% formic acid Wash2: 0.1%formic acid in water Mass Spectrometer Instrument: PE SCIEX API 4000Interface: Turbo Ionspray Mode: Multiple reaction monitoring Method:1.75 minute duration Settings:

TABLE 13-2 Mass Spectrometer Settings Test Article Q1/Q3 DP EP CE CXP ISTEM CAD CUR GS1 GS2 I-12 +1042.9/282.4 175 10 49 18 5500 500 7 30 50 50+1059.9/282.4 60 10 62 18 5500 500 7 30 50 50  +1059.9/1043.0 60 10 2937 5500 500 7 30 50 50 PEA +300.3/62.1 123 10 29 10 5500 500 7 30 50 50

Example 14: PEA Stability in Simulated Intestinal Fluid SimulatedIntestinal Fluid Stability

Studies were carried out in simulated intestinal fluid containingpancreatin. Simulated intestinal fluid was prepared by dissolving 6.8 gof monobasic potassium phosphate in 1.0 L of water. Pancreatin was thenadded to the solution and the pH was adjusted to 6.8. A DMSO stock wasfirst prepared for test articles. Aliquots of DMSO solution were dosedinto 300 μL of matrix, which had been pre-warmed to 37° C., at a finaltest article concentration of 1 μM. An individual tube was dosed foreach time point. At each time point (0, 15, 30, 60, and 120 minutes),900 μL of ice-cold acetonitrile containing 1.0% formic acid was added toan individual tube. The starting time for each tube was staggered suchthat all time points would finish at the same time. After the conclusionof the experiment, tubes were mixed and then centrifuged at 3,000 rpmfor 10 minutes. Calibration standards were prepared in matched matrix.Samples and standards were assayed by LC-MS/MS using electrosprayionization. Analytical conditions are outlined in Appendix 14-1. Testarticle concentration at each time point was compared to test articleconcentration at time 0 to determine the percent remaining at each timepoint. Halflives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 14aand 14b.

TABLE 14a PEA stability observed in simulated intestinal fluid (SIF). %Remaining of Initial (n = 1) Half- Test 15 30 60 120 life^(a) ArticleTreatment 0 min min min min min (min) I-8 SIF + Pancreatin 100 17 6.8<1.3 <1.3 6.2 I-16 SIF + Pancreatin 100 <4.5 <4.5 <4.5 <4.5 ND

TABLE 14b Measured Concentrations of Drug. Concentration (μM) Test 15 3060 120 Article Treatment 0 min min min min min I-8 SIF + Pancreatin 0.940.16 0.064 0 0 I-16 SIF + Pancreatin 0.088 0 0 0 0

Appendix 14-1

Column: Waters ACQUITY UPLC BEH C18 30 × 2.1 mm, 1.7 μm M.P. Buffer: 25mM ammonium formate buffer,, pH 3.5 Aqueous Reservoir (A): 90% water,10% buffer Organic Reservoir (B): 90% acetonitrile, 10% buffer FlowRate: 0.7 mL/minute Gradient Program:

Time (mm) % A % B 0.0 50 50 .75 1 99 1.00 1 99 1.05 50 50 1.50 50 50Total Run Time: 1.5 minutes Autosampler: 1 μL injection volume Wash1:water/methanol/2-propanol: 1/1/1; with 0.2% formic acid Wash2: 0.1%formic acid in water Mass Spectrometer Instrument: PE SCIEX API 4000Interface: Turbo Ionspray Mode: Multiple reaction monitoring Method: 1.5minute duration Settings:

TABLE 14-2 Mass Spectrometer Settings Test Article Q1/Q3 DP EP CE CXP ISTEM CAD CUR GS1 GS2 I-8 +474.3/282.2 112 10 28 18 5500 500 7 30 50 50I-16 +614.5/282.6 123 10 37 18 5500 500 7 30 50 50

Example 15: Determination of the Bioavailability ofPalmitoylethanolamide (PEA) Following Oral Administration of PEA-Prodrugin Male Sprague-Dawley Rats

The present Example describes oral bioavailability of PEA followingadministration of PEA prodrugs in male Sprague-Dawley rats.

Oral bioavailability of palmitoylethanolamide (PEA) was evaluated inmale Sprague-Dawley rats following oral administration of PEA pro-drugs,I-15 and I-14. Each test article was dosed orally (PO) at 20.7 mg/kg,which was equivalent to a 10 mg/kg dose of PEA. Blood samples werecollected up to 8 hours post-dose, and plasma concentrations of PEA weredetermined by LC-MS/MS. Following PO dosing of I-15 (in 20% (SolutolHS15:NMP 1:1) 10% PEG400; 70% H₂O), average C_(max) of 11.7±2.34 ng/mLwas observed between 15 minutes and 1 hour post dose. Average exposurefor I-15 (Group 1) based on the dose-normalized AUC_(last) was 2.13±1.05hr*kg*ng/mL/mg. Based on the IV data from Example 5, average oralbioavailability for I-15 (Group 1) was 3.42±1.69%. Following PO dosingof I-14 (in 20% (Solutol HS15:NMP 1:1) 10% PEG400; 70% H₂O), averageC_(max) of 16.9±1.47 ng/mL was observed at 30 minutes post dose in allrats. Average exposure for I-14 (Group 2) based on dose-normalizedAUC_(last) was 2.72±0.854 hr*kg*ng/mL/mg. Based on the IV data fromExample 5, average oral bioavailability for I-14 (Group 2) was4.39±1.37%.

Preparation of Dosing Formulations

Pro-drugs were dosed so that a total dose of 10 mg/kg of PEA wasadministered. Prodrugs were formulated in a vehicle comprised of 10%Solutol HS15, 10% n-methyl pyrrolidone (NMP), 10% polyethylene glycol400 (PEG400) and 70% water.

Animal Dosing

Pharmacokinetics of PEA was evaluated in fasted male Sprague-Dawleyrats. Rats were housed one per cage. Each rat was fitted with a jugularvein cannula (JVC) for blood collection. Each study group was dosing intriplicate. Rats were fasted for a minimum of twelve hours prior todosing. Food was returned at four hours post dosing. Animals had freeaccess to water throughout the study. Blood samples (˜300 μL) werecollected from the rats via a JVC and placed into chilled polypropylenetubes containing sodium heparin as an anticoagulant, and 30 μL of 0.5 Mcitric acid. Samples were maintained chilled throughout processing.Blood samples were centrifuged at 4° C. and 3,000 g for 5 minutes.Plasma (˜150 μL) was then transferred to a chilled, labeledpolypropylene tube containing 15 μL of 10% formic acid, placed on dryice, and stored in a freezer maintained at −60° C. to −80° C. Bloodsampling times are shown in Table 15a.

TABLE 15a Study Design. Dose Dosing Blood Total (mg/kg Solution DosingSample Dose Test Dosing Animals of pro- Conc. Volume Time group ArticleRoute n = drug)* (mg/mL) (mL/kg) Vehicle Points 1 I-15 PO 3 20.7 3 6.920% Pre-dose, (Solutol 5, 15, HS15:NMP 30 min, 1:1 10% 1, 2, PEG400; 4,8 hours 70% H₂O 2 I-14 PO 3 20.7 3 6.9 20% Predose, (Solutol 5, 15,HS15:NMP 30 min, 1:1) 10% 1, 2, PEG400; 4, 8 hours 70% H₂O *All dosesare based on mg/kg of the pro-drugs, and deliver 10 mg/kg of activedrug, PEA.

An LC-MS/MS method for the determination of PEA and PEA-prodrug isdescribed above (see e.g., Example 3).

Pharmacokinetic parameters were calculated from the time course of theplasma concentration. Maximum plasma concentration (C_(max)) and thetime to reach maximum plasma drug concentration (T_(max)) after oraldosing were observed from data. Area under the time concentration curve(AUC) was calculated using the linear trapezoidal rule with calculationto the last quantifiable data point, and with extrapolation to infinityif applicable. At least three quantifiable data points were required todetermine the AUC. Plasma half-life (t_(1/2)) was calculated from0.693/slope of the terminal elimination phase. Mean residence time, MRT,was calculated by dividing area under the moment curve (AUMC) by AUC.Bioavailability was determined by dividing individual dose-normalized POAUC_(last) values by the average IV AUC_(last) value (IV data fromExample 5). Samples below the limit of quantitation were treated as zerofor pharmacokinetic data analysis.

Results

No adverse reactions were observed following the oral administration ofPEA pro-drug in male Sprague-Dawley rats in this study. Dosing solutionswere not analyzed by LC-MS/MS. Nominal dosing level was used in allcalculations. Individual and average plasma concentrations for PEA andare shown in Table 15b and Table 15c. Data are expressed as ng/mL of thefree drug. Samples that were below the limit of quantitation were notused in the calculation of averages. Plasma concentrations versus timedata are plotted in FIGS. 15A through 15D. Endogenous levels of PEAfound in all rats were below the limit of quantitation; and therefore,measured concentrations of PEA in plasma samples were not corrected.

TABLE 15b Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-15 in20% Solutol HS 15:NMP (1:1), 10% PEG400, 70% H₂O) at 20.7 mg/kg in MaleSprague-Dawley Rats (Group 1) Oral (20.7 mg/kg I-15 equals 10 mg/kg PEA)Rat # Time (hr) 305 306 307 Mean SD 0 (pre-dose) BLOQ BLOQ BLOQ ND ND0.083 BLOQ 3.92 BLOQ 3.92 ND 0.25 8.62 9.44 9.00 9.02  0.410 0.50 11.4 12.6  8.92 11.0  1.88 1.0 13.4  6.95 4.60 8.32 4.56 2.0 9.93 10.7  4.368.33 3.46 4.0 BLOQ 3.41 BLOQ ND ND 8.0 BLOQ BLOQ BLOQ ND ND AnimalWeight (kg)  0.250  0.235  0.247  0.244  0.008 Volume Dosed (mL) 1.731.62 1.70 1.68 0.06 C_(max) (ng/mL) 13.4  12.6  9.00 11.7  2.34 t_(max)(hr) 1.0  0.50 0.25 0.58 0.38 t_(1/2)(hr) ND³ ND⁴ ND⁴ ND ND MRT_(last)(hr) 1.07 1.69  0.917 1.23  0.409 AUC_(last) (hr ng/mL) 21.1  31.9 10.9  21.3  10.5  AUC_(∞) (hr ng/mL) ND³ ND⁴ ND⁴ ND ND Dose-normalizedValues¹ AUC_(last) (hr kg 2.11 3.19 1.09 2.13 1.05 ng/mL/mg) AUC_(∞) (hrkg ng/mL/mg) ND³ ND⁴ ND⁴ ND ND Bioavailability (%)² 3.40 5.13 1.75 3.421.69 C_(max): maximum plasma concentration; t_(max): time of maximumplasma concentration; t_(1/2): half-life, data points used for half-lifedetermination are in bold; MRT_(last): mean residence time, calculatedto the last observable time point; AUC_(last): area under the curve,calculated to the last observable time point; AUC_(∞): area under thecurve, extrapolated to infinity; ND: not determined; BLOQ: below thelimit of quantitation (2.5 ng/mL); ¹Dose-normalized by dividing theparameter by nominal dose in mg/kg; ²Bioavailability determined bydividing individual dose-normalized oral AUC_(last) values by theaverage IV AUC_(last) value 62.1 hr*ng/mL from Example 5; ³Notdetermined due to lack of quantifiable data points trailing the C_(max).⁴Not determined because the line defining the terminal elimination phasehad an r² >0.85.

TABLE 15b Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-14 in20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 20.7 mg/kg in MaleSprague-Dawley Rats (Group 1) Oral (20.7 mg/kg I-14 equals 10 mg/kg PEA)Rat # Time (hr) 308 309 310 Mean SD 0 (pre-dose) BLOQ BLOQ BLOQ ND ND0.083 4.46 BLOQ 4.54 4.50 ND 0.25 11.3  4.91 14.1 10.1  4.71 0.50 15.8 16.4  18.6 16.9  1.47 1.0 14.8  13.7  11.0 13.2  1.96 2.0 9.02 5.99 9.418.14 1.87 4.0 BLOQ BLOQ 3.97 ND ND 8.0 BLOQ BLOQ BLOQ ND ND AnimalWeight (kg)  0.239  0.248 0.249  0.245  0.006 Volume Dosed (mL) 1.651.71 1.72 1.69 0.04 C_(max) (ng/mL) 15.8  16.4  18.6 16.9  1.47 t_(max)(hr) 0.50 0.50 0.50 0.50 0.00 t_(1/2)(hr) ND³ ND³ 1.96 ND ND MRT_(last)(hr)  0.970  0.959 1.54 1.16  0.331 AUC_(last) (hr ng/mL) 24.4  20.4 36.8 27.2  8.54 AUC_(∞) (hr ng/mL) ND³ ND³ 48.1 ND ND Dose-normalizedValues¹ AUC_(last) (hr kg 2.44 2.04 3.68 2.72  0.854 ng/mL/mg) AUC_(∞)(hr kg ng/mL/mg) ND³ ND³ 4.81 ND ND Bioavailability (%)² 3.94 3.29 5.934.39 1.37 C_(max): maximum plasma concentration; t_(max): time ofmaximum plasma concentration; t_(1/2): half-life, data points used forhalf-life determination are in bold; MRT_(last): mean residence time,calculated to the last observable time point; AUC_(last): area under thecurve, calculated to the last observable time point; AUC_(∞): area underthe curve, extrapolated to infinity; ND: not determined; BLOQ: below thelimit of quantitation (2.5 ng/mL); ¹Dose-normalized by dividing theparameter by nominal dose in mg/kg; ²Bioavailability determined bydividing individual dose-normalized oral AUC_(last) values by theaverage IV AUC_(last) value 62.1 hr*ng/mL from Example 5; ³Notdetermined due to lack of quantifiable data points trailing the C_(max).

Example 16: Determined of Bioavailability of Palmitoylethanolamide (PEA)Following Oral Administration of PEA-Prodrug in Male Sprague-Dawley Rats

The present Example describes oral bioavailability of PEA followingadministration of PEA prodrugs I-8 and I-16 in male Sprague-Dawley ratsaccording to the methods described in, e.g., Example 15. Individual andaverage plasma concentrations for PEA and are shown in Table 16a andTable 16b. Plasma concentrations versus time data are plotted in FIGS.9A through 9D.

TABLE 16a Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-8 in20% Solutol HS15:NMP (1:1). 10% PEG400, 70% H₂O) at 16 mg/kg in MaleSprague-Dawley Rats (Group 1). Oral (16 mg/kg I-8 equals 10 mg/kg PEA)Rat # Time (hr) 317 318 319 Mean SD 0 BLOQ BLOQ BLOQ ND ND 0.083 3.063.99 4.73 3.93 0.837 0.25 11.2 8.54 11.4 10.4 1.60 0.50 52.5 33.1 42.942.8 9.70 1.0 65.1 41.4 65.0 57.2 13.7 2.0 29.2 26.9 21.8 26.0 3.79 4.08.08 6.40 7.22 7.23 0.840 8.0 BLOQ 2.50 BLOQ ND ND Animal Weight (kg)0.275 0.269 0.271 0.272 0.003 Volume Dosed (mL) 1.46 1.43 1.44 1.44 0.02C_(max) (ng/mL) 65.1 41.4 65.0 57.2 13.7 t_(max) (hr) 1.0 1.0 1.0 1.00.0 t_(1/2)(hr) ND³ 1.86 ND³ ND ND MRT_(last) (hr) 1.56 2.23 1.49 1.760.411 AUC_(last) (hr ng/mL) 114 105 103 107 6.17 AUC_(∞) (hr ng/mL) ND³112 ND³ ND ND Dose-normalized Values¹ AUC_(last) (hr kg 11.4 10.5 10.310.7 0.617 ng/mL/mg) AUC_(∞) (hr kg ng/mL/mg) ND³ 11.2 ND³ ND NDBioavailability (%)² 18.4 16.9 16.5 17.3 0.994 C_(max): maximum plasmaconcentration; t_(max): time of maximum plasma concentration; t_(1/2):half-life, data points used for half-life determination are in bold;MRT_(last): mean residence time, calculated to the last observable timepoint; AUC_(last): area under the curve, calculated to the lastobservable time point; AUC_(∞): area under the curve, extrapolated toinfinity; ND: not determined; BLOQ: below the limit of quantitation (2.5ng/mL); ¹Dose-normalized by dividing the parameter by nominal dose inmg/kg; ²Bioavailability determined by dividing individualdose-normalized oral AUC_(last) values by the average IV AUC_(last)value 62.1 hr*ng/mL from Example 5; ³Not determined due to lack ofquantifiable data points trailing the C_(max).

TABLE 16b Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-16 in20% Solutol HS15:NMP (1:1), 10% PEG400. 70% H₂O) at 16 mg/kg in MaleSprague-Dawley Rats (Group 1). Oral (16 mg/kg I-16 equals 10 mg/kg PEA)Rat # Time (hr) 320 321 322 Mean SD 0 BLOQ BLOQ BLOQ ND ND 0.083 2.792.93 BLOQ 2.86 ND 0.25 8.32 5.06 4.65 6.01 2.01 0.50 43.4 23.0  18.5 28.3  13.3  1.0 55.0 34.3  40.2  43.2  10.7  2.0 27.8 14.0  12.8  18.2 8.34 4.0 6.81 8.79 5.15 6.92 1.82 8.0 BLOQ BLOQ BLOQ ND ND Animal Weight(kg) 0.279  0.269  0.278  0.275  0.006 Volume Dosed (mL) 1.93 1.86 1.921.90 0.04 C_(max) (ng/mL) 55.0 34.3  40.2  43.2  10.7  t_(max) (hr) 1.01.0  1.0  1.0  0.0  t_(1/2)(hr) ND³ ND³ ND³ ND ND MRT_(last) (hr) 1.581.72 1.54 1.62  0.0980 AUC_(last) (hr ng/mL) 101 62.5  61.7  75.0  22.4 AUC_(∞) (hr ng/mL) ND³ ND³ ND³ ND ND Dose-normalized Values¹ AUC_(last)(hr kg 10.1 6.25 6.17 7.50 2.24 ng/mL/mg) AUC_(∞) (hr kg ND³ ND³ ND³ NDND ng/mL/mg) Bioavailability (%)² 16.2 10.1  9.93 12.1  3.60 C_(max):maximum plasma concentration; t_(max): time of maximum plasmaconcentration; t_(1/2): half-life, data points used for half-lifedetermination are in bold; MRT_(last): mean residence time, calculatedto the last observable time point; AUC_(last): area under the curve,calculated to the last observable time point; AUC_(∞): area under thecurve, extrapolated to infinity; ND: not determined; BLOQ: below thelimit of quantitation (2.5 ng/mL); ¹Dose-normalized by dividing theparameter by nominal dose in mg/kg; ²Bioavailability determined bydividing individual dose-normalized oral AUC_(last) values by theaverage IV AUC_(last) value 62.1 hr*ng/mL from Example 5; ³Notdetermined due to lack of quantifiable data points trailing the C_(max).

Example 17: PEA Stability in Human, Rat, Mouse and Dog Liver Microsomes,Human, Rat, Mouse and Dog Liver S9 Fraction, Human, Rate, Mouse and DogIntestinal S9 Fraction, Human Rat, Mouse and Dog Plasma, and SimulatedIntestinal Fluid

The present Example describes PEA stability observed in 1) human, rat,mouse, and dog liver microsomes; 2) human, rat, mouse, dog liver S9fraction; human, rat, mouse, and dog intestinal S9 fraction; 4) human,rat, mouse, and dog plasma; and 5) simulated intestinal fluid containingvarious enzymes.

Liver Microsomal Stability

Mixed-gender human (Lot #1010420), male Sprague-Dawley rat (Lot#1510115), male CD-1 mouse (Lot #1610148), and male Beagle dog (Lot#1110044) liver microsomes were provided. Reaction mixture, minuscofactors, was prepared as described below. Test article was added intothe reaction mixture at a final concentration of 1 μM. Control compound,testosterone, was run simultaneously with the test article in a separatereaction. An aliquot of the reaction mixture (without cofactor) wasequilibrated in a shaking water bath at 37° C. for 3 minutes. Reactionwas initiated by the addition of cofactor, and the mixture was incubatedin a shaking water bath at 37° C. Aliquots (100 μL) were withdrawn at 0,10, 20, 30, and 60 minutes. Test article samples were immediatelycombined with 300 μL, of ice-cold acetonitrile containing 1% formicacid. Control samples were immediately combined with 400 μL of ice-cold50/50 acetonitrile (ACN)/dH₂O containing 0.1% formic acid and internalstandard to terminate the reaction. Samples were then mixed andcentrifuged to precipitate proteins. Calibration standards were preparedin matched matrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both dosed prodnig and expected drug (PEA).Analytical conditions are outlined in Appendix 17-1. Test articleconcentration at each time point was compared to test articleconcentration at time 0 to determine the percent remaining at each timepoint. Half-lives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 17aand 17b.

Reaction Composition

Liver Microsomes 0.5 mg/mL NADPH (cofactor) 1 mM Potassium Phosphate pH7.4 100 mM Magnesium Chloride 5 mM Test Article 1 μL

TABLE 17a PEA stability observed in human, rat, mouse, and dog livermicrosomes. CL_(int) ^(b) % Remaining of Initial (n = 1) Half- (mL/min/Test 10 20 30 60 life^(a) mg Article Species 0 min min min min min (min)protein) I-8 Human 100 2.8 <1.0 <1.0 <1.0 <10 (2.0) >0.139 (0.712) Rat100 4.3 1.2 <1.0 <1.0 <10 (2.2) >0.139 (0.624) Mouse 100 <1.0 <1.0 <1.0<1.0 <1.0 >1.38 Dog 100 <1.0 <1.0 <1.0 <1.0 <1.0 >1.38 I-16 Human 1008.0 3.5 1.9 <1.0 <10 (2.8) >0.139 (0.493) Rat 100 9.7 4.2 3.1 <1.0 <10(3.1) >0.139 (0.452) Mouse 100 7.6 3.0 2.1 1.8 <10 (2.8) >0.139 (0.503)Dog 100 4.4 1.6 1.4 1.5 <10 (2.2) >0.139 (0.618) ^(a)When the calculatedhalf-life is longer than the duration of the experiment, half-life isexpressed as > the longest incubation time. Similarly, if calculatedhalf-life is less than the shortest time point, half-life is expressedas < that time point and calculated half-life is also listed inparentheses. ^(b)Intrinsic clearance (CL_(int)) was calculated based onCL_(int) = k/P, where k is elimination rate constant and P is proteinconcentration in the incubation.

Half- CL_(int) Acceptable Control life (ml/min/mg Range Compound Species(min) protein) (t_(1/2), min) Testosterone Human 9.2 0.151 ≤41 Rat 1.50.911 ≤15 Mouse 2.1 0.674 ≤15 Dog 23 0.0593 ≤40

TABLE 17b Measured Concentrations of Drug. Dosed Concentration (μM) Test10 20 30 60 Article Species Analyte 0 min min min min min I-8 Human PEA0.063 0.34 0.25 0.21 0.070 Rat 0.033 0.041 0.019 0 0 Mouse 0.45 0.300.15 0.086 0 Dog 0.33 0.54 0.46 0.44 0.20 I-16 Human 0 0.22 0.15 0.140.069 Rat 0.025 0.033 0.015 0 0 Mouse 0.29 0.27 0.12 0.089 0.0093 Dog0.053 0.59 0.48 0.43 0.20

Liver S9 Stability

Mixed gender human (Lot #0910396), male Sprague-Dawley rat (Lot#1410265), male CD-1 mouse (Lot #1310026), and male Beagle dog (Lot#1310285) liver S9 fraction were provided. Reaction mixture, minuscofactors, was prepared as described below. Test article was added intothe reaction mixture at a final concentration of 1 μM. Controlcompounds, testosterone and 7-hydroxycoumarin (7-HC), were runsimultaneously with the test article in a separate reaction. An aliquotof the reaction mixture (without cofactor cocktail) was equilibrated ina shaking water bath at 37° C. for 3 minutes. Reaction was initiated bythe addition of cofactor cocktail (see below), and the mixture was thenincubated in a shaking water bath at 37° C. Aliquots (100 μL) werewithdrawn at 0, 10, 20, 30, and 60 minutes. Test article samples wereimmediately combined with 300 μL of ice-cold acetonitrile containing 1%formic acid. Control samples were immediately combined with 400 μL ofice-cold 50/50 acetonitrile (ACN)/dH₂O containing 0.1% formic acid andinternal standard to terminate the reaction. Samples were then mixed andcentrifuged to precipitate proteins. Calibration standards were preparedin matched matrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both dosed prodrug and the expected drug(PEA). Analytical conditions are outlined in Appendix 17-1. Test articleconcentration at each time point was compared to test articleconcentration at time 0 to determine the percent remaining at each timepoint. Half-lives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 17cand 17d.

Reset Composition

Liver S9 Fraction 1.0 mg/mL NADPH (cofactor) 1 mM UDPGA (cofactor) 1 mMPAPS (cofactor) 1 mM GSH (cofactor) 1 mM Potassium Phosphate pH 7.4 100mM Magnesium Chloride 5 mM Test Article 1 μM

TABLE 17c PEA stability observed in human, rat, mouse, and dog liver S9.CL_(int) ^(b) % Remaining of Initial (n = 1) Half- (mL/min/ Test 10 2030 60 life^(a) mg Article Species 0 min min min min min (min) protein)I-8 Human 100 1.4 <1.0 <1.0 <1.0 <10 (1.6) >0.0693 (0.431) Rat 100 172.8 <1.0 <1.0 <10 (3.9) >0.0693 (0.177) Mouse 100 4.6 1.0 <1.0 <1.0 <10(2.3) >0.0693 (0.305) Dog 100 32 11 3.5 <1.0 <10 (6.1) >0.0693 (0.113)I-16 Human 100 4.9 2.1 <1.0 <1.0 <10 (2.3) >0.0693 (0.299) Rat 100 3.0<1.0 <1.0 <1.0 <10 (2.0) >0.0693 (0.352) Mouse 100 3.7 <1.0 <1.0 <1.0<10 (2.1) >0.0693 (0.330) Dog 100 2.1 <1.0 <1.0 <1.0 <10 (1.8) >0.0693(0.385) ^(a)When the calculated half-life is longer than the duration ofthe experiment, half-life is expressed as > the longest incubation time.Similarly, if calculated half-life is less than the shortest time point,half-life is expressed as < that time point and calculated half-life isalso listed in parentheses. ^(b)Intrinsic clearance (CL_(int)) wascalculated based on CL_(int) = k/P, where k is the elimination rateconstant and P is the protein concentration in the incubation.

Half- CL_(int) Acceptable life (ml/min/mg Range Control Compound Species(min) protein) (t_(1/2), min) Testosterone Human 15 0.0463 ≤34 Rat 2.70.260 ≤15 Mouse 17 0.0417 ≤37 Dog 25 0.0272 ≤42 7-hydroxycourmarin Human11 0.0628 <18 Rat 2.5 0.283 <15 Mouse 4.3 0.162 <15 Dog 2.1 0.334 <15

TABLE 17d Measured Concentrations of Drug. Dosed Concentration (μM) Test10 20 30 60 Article Species Analyte 0 min min min min min I-8 Human PEA0.19 0.57 0.47 0.41 0.28 Rat 0.069 0.17 0.092 0.061 0.013 Mouse 0.290.50 0.44 0.38 0.37 Dog 0.13 0.33 0.34 0.33 0.20 I-16 Human 0.10 0.460.42 0.39 0.27 Rat 0.050 0.13 0.13 0.058 0.020 Mouse 0.33 0.78 0.68 0.590.43 Dog 0.053 0.26 0.33 0.29 0.23

Intestinal S9 Fraction Stability

Mixed-gender human (Lot #1410073), male Sprague-Dawley rat (Lot#1510303), male CD-1 mouse (Lot #1510194), and male Beagle dog (Lot#1510226) intestinal S9 fraction were provided. Reaction mixture, minuscofactors, was prepared as described below. Test article was added intothe reaction mixture at a final concentration of 1 μM. Controlcompounds, testosterone and 7-hydroxycoumarin, were run simultaneouslywith the test article in a separate reaction. An aliquot of the reactionmixture (without cofactor cocktail) was equilibrated in a shaking waterbath at 37° C. for 3 minutes. Reaction was initiated by the addition ofcofactor cocktail, and the mixture was incubated in a shaking water bathat 37° C. Aliquots (100 μL) were withdrawn at 0, 10, 20, 30, and 60minutes. Test article samples were immediately combined with 300 μL ofice-cold acetonitrile containing 1% formic acid. Control samples wereimmediately combined with 400 μL of ice-cold 50/50 acetonitrile(ACN)/dH₂O containing 0.1% formic acid and internal standard toterminate the reaction. Samples were then mixed and centrifuged toprecipitate proteins. Calibration standards were prepared in matchedmatrix. Samples and standards were assayed by LC-MS/MS usingelectrospray ionization for both dosed prodrug and expected drug (PEA).Analytical conditions are outlined in Appendix 17-1. Test articleconcentration at each time point was compared to test articleconcentration at time 0 to determine the percent remaining at each timepoint. Halflives were calculated using GraphPad software, fitting to asingle-phase exponential decay equation. Results are shown in Tables 17eand 17f.

Reaction Compostion

Intestinal S9 Fraction 1.0 mg/mL NADPH (cofactor) 1 mM UDPGA (cofactor)1 mM PAPS (cofactor) 1 mM GSH (cofactor) 1 mM Potassium Phosphate, pH7.4 100 mM Magnesium Chloride 5 mM Test Article 1 μM

TABLE 17e PEA stability observed in human, rat, mouse, and dogintestinal S9 fraction. CL_(int) ^(b) % Remaining of Initial (n = 1)Half- (mL/min/ Test 10 20 30 60 life^(a) mg Article Species 0 min mm minmin min (min) protein) I-8 Human 100 31 7.0 1.8 <1.0 <10 (5.7) >0.0693 (0.122) Rat 100 58 37 22 6.2 14 0.0503 Mouse 100 65 45 31 12 18 0.0389Dog 100 <1.0 <1.0 <1.0 <1.0 <10 (1.5) >0.0693  (0.473) I-16 Human 1003.2 <1.0 <1.0 <1.0 <10 (2.0) >0.0693  (0.344) Rat 100 <1.0 <1.0 <1.0<1.0  <1.0 >0.691    Mouse 100 2.0 <1.0 <1.0 <1.0 <10 (1.8) >0.0693 (0.389) Dog 100 1.7 <1.0 <1.0 <1.0 <10 (1.7) >0.0693  (0.409) ^(a)Whencalculated half-life is longer than the duration of the experiment,half-life is expressed as > the longest incubation time. Similarly, ifcalculated half-life is less than shortest time point, half-life isexpressed as < that time point and calculated half-life is also listedin parentheses. ^(b)Intrinsic clearance (CL_(int)) was calculated basedon CL_(int) = k/P, where k is elimination rate constant and P is proteinconcentration in the incubation.

CL_(in) Control Half-life (ml/min/mg Compound Species (min) protein)Testosterone Human 4.8 0.144 Rat >60 (102) <0.0116 (0.00680) Mouse >60(90)  <0.0116 (0.00767) Dog >60 <0.0116 7-hydroxycourmarin Human 130.0522 Rat 29 0.0242 Mouse 5.0 0.138 Dog 9.0 0.0770

TABLE 17f Measured Concentrations of Drug. Dosed Concentration (μM) Test10 20 30 60 Article Species Analyte 0 min min min min min I-8 Human PEA0.021 0.32 0.39 0.38 0.38 Rat 0.022 0.22 0.34 0.42 0.45 Mouse 0.012 0.120.19 0.22 0.26 Dog 0.33 0.71 0.81 0.67 0.81 I-16 Human 0 0.17 0.29 0.330.32 Rat 0 0.20 0.28 0.34 0.38 Mouse 0 0.10 0.17 0.18 0.26 Dog 0.0420.50 0.57 0.54 0.68

Plasma Stability

Studies were carried out in mixed-gender human plasma (Lot #AS 1650-2),male Sprague-Dawley rat (Lot #RAT320835), male CD-1 mouse (Lot#MSE260693), and 6 male Beagle dog (Lot #BGL91384), on sodium heparin.Plasma was adjusted to pH 7.4 prior to initiating the experiments. DMSOstocks were first prepared for the test articles. Aliquots of the DMSOsolutions were dosed into 700 μL of plasma, which had been pre-warmed to37° C., at a final test article concentration of 1 μM. Aliquots (100 μL)were taken at each time point (0, 15, 30, 60, and 120 minutes) and wereimmediately combined with 300 μL of ice-cold acetonitrile containing 1%formic acid. Samples were stored at 4° C. until the end of theexperiment. After the final time point was sampled, the plate was mixedand then centrifuged at 3,000 rpm for 10 minutes. Calibration standardswere prepared in matched matrix. Samples and standards were assayed byLC-MS/MS using electrospray ionization for both dosed prodrug andexpected drug (PEA). Analytical conditions are outlined in Appendix17-1. Test article concentration at each time point was compared to testarticle concentration at time 0 to determine the percent remaining ateach time point. Half-lives were calculated using GraphPad software,fitting to a single-phase exponential decay equation. Results are shownin Tables 17g and 17h.

TABLE 17g PEA stability observed in human, rat mouse, and dog plasma. %Remaining of Initial (n = 1) Half- Test 15 30 60 120 life^(a) ArticleSpecies 0 min min min min min (min) I-8 Human 100 <1.0 <1.0 <1.0 <1.0<1.0 Rat 100 <1.0 <1.0 <1.0 <1.0 <1.0 Mouse 100 <1.0 <1.0 <1.0 <1.0 <1.0Dog 100 <1.0 <1.0 <1.0 <1.0 <1.0 I-16 Human 100 78 51 24 5.8 31 Rat 10020 9.3 4.1 1.6 <15 (6.9) Mouse 100 10 4.0  1.1 <1.0 <15 (4.7) Dog 100 9165 39 13 44

TABLE 17h Measured Concentrations of Drug. Dosed Concentration (μM) Test15 30 60 120 Article Species Analyte 0 min min min min min I-8 Human PEA0.27 1.25 1.30 1.16 1.50 Rat 0.21 0.62 0.64 0.55 0.59 Mouse 0.97 0.930.92 1.05 1.30 Dog 0.33 0.72 0.72 0.73 0.69 I-16 Human 0 0.19 0.38 0.530.67 Rat 0 0.23 0.24 0.24 0.30 Mouse 0.12 0.53 0.55 0.68 0.79 Dog 0 0.120.18 0.34 0.44

Simulated Intestinal Fluid Stability

Studies were carried out in simulated intestinal fluid in the presenceof various enzymes. Simulated intestinal fluid was prepared bydissolving 6.8 g of monobasic potassium phosphate in 1.0 L of water.Aliquots of this solution were taken and the pH was adjusted to 6.8.Individual enzymes were then spiked into aliquots for each experiment. ADMSO stock was first prepared for the test article. Aliquots of the DMSOsolution were dosed into 700 μL of matrix, which had been pre-warmed to37° C., at a final test article concentration of 1 μM. Aliquots (100 μL)were taken at each time point (0, 15, 30, 60, and 120 minutes) and wereimmediately combined with 300 μL of ice-cold acetonitrile containing 1%formic acid. Samples were stored at 4° C. until the end of theexperiment. After the final time point was sampled, the plate was mixedand then centrifuged at 3,000 rpm for 10 minutes. Calibration standardswere prepared in matched matrix. Samples and standards were assayed byLC-MS/MS using electrospray ionization for both dosed prodrug andexpected drug (PEA). Analytical conditions are outlined in Appendix17-1. Test article concentration at each time point was compared to thetest article concentration at time 0 to determine the percent remainingat each time point. Half-lives were calculated using GraphPad software,fitting to a single-phase exponential decay equation. Results are shownin Tables 17i and 17j.

TABLE 17i PEA stability observed in simulated intestinal fluid (SIF). %Remaining of Initial (n = 1) Half- Test 15 30 60 120 life^(a) ArticleTreatment 0 min min min min min (min) I-8 SIF + Elastase 100 38 17 16 17<15 (12)  SIF + Carboxypeptidase A 100 33 8.7 <1.0 <1.0 <15 (9.2) SIF +Carboxypeptidase B 100 30 7.1 <1.0 <1.0 <15 (8.5) SIF + Chymotrypsin 10036 8.7 <1.0 <1.0 <15 (9.6) SIF + Trypsin 100 52 31 28 25 25 I-16 SIF +Elastase 100 72 50 27 7.1 31 SIF + Carboxypeptidase A 100 88 74 60 32 75SIF + Carboxypeptidase B 100 94 87 68 37 89 SIF + Chymotrypsin 100 10390 68 35 84 SIF + Trypsin 100 89 82 62 37 85 ^(a)When the calculatedhalf-life is longer than the duration of the experiment, half-life isexpressed as > the longest incubation time. Similarly, if calculatedhalf-life is less than the shortest time point, half-life is expressedas < that time point and calculated half-life is also listed inparentheses.

TABLE 17j Measured Concentrations of Drug. Concentration (μM) Test 15 3060 120 Article Treatment Analyte 0 min mm min min min I-8 SIF + ElastasePEA 0 0 0 0 0 SIF + Carboxypeptidase A 0 0 0 0 0 SIF + CarboxypeptidaseB 0 0 0 0 0 SIF + Chymotrypsin 0 0 0 0 0 SIF + Trypsin 0 0 0 0 0 I-16SIF + Elastase 0 0 0 0 0 SIF + Carboxypeptidase A 0 0 0 0 0 SIF +Carboxypeptidase B 0 0 0 0 0 SIF + Chymotrypsin 0 0 0 0 0 SIF + Trypsin0 0 0 0 0

Appendix 17-1 Liquid Chromatography

Column: Waters ACQUITY UPLC BEH Phenyl 30 × 2.1 mm, 1.7 μm M.P. Buffer:25 mM ammonium formate buffer,, pH 3.5 Aqueous Reservoir (A): 90% water,10% buffer Organic Reservoir (B): 90% acetonitrile, 10% buffer FlowRate: 0.7 mL/minute Gradient Program:

TABLE 17-1 Liquid Chromatography Gradient Program Time (mm) % A % B 0.050 50 2.00 15 85 2.05 50 50 2.50 50 50 Total Run Time: 2.5 minutesAutosampler: 3 μL injection volume Wash1: water/methanol/2-propanol:1/1/1; with 0.2% formic acid Wash2: 0.1% formic acid in water MassSpectrometer Instrument: PE SCIEX API 4000 Interface: Turbo IonsprayMode: Multiple reaction monitoring Method: 2.5 minute duration Settings:

TABLE 17-2 Mass Spectrometer Settings Test Article +/− Q1 Q3 DP EP CECXP IS I-8 + 474.3 282.2 112 10 28 18 5500 I-16 + 614.5 282.6 123 10 3718 5500 PEA + 300.3 62.0 100 10 32 10 5500

TABLE A Summary of Half Life and Oral Bioavailability Data. CompoundHalf life (mins) Bioavailability PEA n/a 0.56% I-11 <1 4.5% I-15 <13.42% I-14 7.5 4.39% I-16 <1 12.1% I-8 6.2 17.3%

Example 18: Determination of the Bioavailability ofPalmitoylethanolamide (PEA) Following Oral Administration of PEA-Prodrugin Male Sprague-Dawley Rats

The present Example describes oral bioavailability of PEA followingadministration of PEA prodrugs in male Sprague-Dawley rats.

Or a the oral bioavailability of palmitoylethanolamide (PEA) wasevaluated in male Sprague-Dawley rats following oral dosing of PEApro-drugs, I-8 and I-16. I-8 was dosed orally (PO) at 4, 8 and 16 mg/kg,and I-16 was dosed orally (PO) at 5.2, 10.35 and 20.7 mg/kg in aformulation consisting of 20% (Solutol HS15:NMP 1:1), 10% PEG400, and70% water. Each prodrug dosed was equivalent to 2.5, 5, or 10 mg/kg doseof PEA. Blood samples were collected up to 8 hours post-dose, and plasmaconcentrations of PEA were determined by LC-MS/MS. Bioavailability wascalculated using IV data from Example 5.

Following PO dosing of I-8 at 4 mg/kg (2.5 mg/kg PEA equivalent) maximumplasma concentrations (average of 25.3±6.67 ng/mL) were observed at 1hour post dosing. Halflife could not be determined due to a lack ofquantifiable data points trailing the C_(max). Average exposure for I-8based on the dose-normalized AUC_(last) was 13.5±4.65 hr*kg*ng/mL/mg.Average oral bioavailability for PEA in this group was 21.7±7.48%.

Following PO dosing of I-8 at 8 mg/kg (5 mg/kg PEA equivalent) maximumplasma concentrations (average of 46.9±13.6 ng/mL) were observed at 1hour post dosing. Halflife could not be determined due to a lack ofquantifiable data points trailing the C_(max). Average exposure for I-8based on the dose-normalized AUC_(last) was 14.8±1.19 hr*kg*ng/mL/mg.Average oral bioavailability for PEA in this group was 23.9±1.92%.

Following PO dosing of I-8 at 16 mg/kg (10 mg/kg PEA equivalent) maximumplasma concentrations (average of 102±31.8 ng/mL) were observed at 1hour post dosing. Half-life could not be determined due to a lack ofquantifiable data points trailing the C_(max). Average exposure for I-8based on the dose-normalized AUC_(last) was 16.8±3.80 hr*kg*ng/mL/mg.Average oral bioavailability for PEA in this group was 27.1±6.13%.

Following PO dosing of I-16 at 5.2 mg/kg (2.5 mg/kg PEA equivalent)maximum plasma concentrations (average of 25.3±23.6 ng/mL) were observedbetween 30 minutes and 1 hour post dosing. Half-life could not bedetermined due to a lack of quantifiable data points trailing theC_(max). Average exposure for I-16 based on the dose-normalizedAUC_(last) was 9.08±6.08 hr*kg*ng/mL/mg. Average oral bioavailabilityfor PEA in this group was 14.6±11.1%.

Following PO dosing of I-16 at 10.35 mg/kg (5 mg/kg PEA equivalent)maximum plasma concentrations (average of 43.9±7.33 ng/mL) were observedat 1 hour post dosing. Halflife could not be determined due to a lack ofquantifiable data points trailing the C_(max). Average exposure for I-16based on the dose-normalized AUC_(last) was 10.6±0.544 hr*kg*ng/mL/mg.Average oral bioavailability for PEA in this group was 17.0±0.876%.

Following PO dosing of I-16 at 20.7 mg/kg (10 mg/kg PEA equivalent)maximum plasma concentrations (average of 68.3±11.4 ng/mL) were observedat 1 hour post dosing. Halflife could not be determined due to a lack ofquantifiable data points trailing the C_(max) Average exposure for I-16based on the dose-normalized AUC_(last) was 11.2±1.01 hr*kg*ng/mL/mg.Average oral bioavailability for PEA in this group was 18.0±1.63%.

Following each dose of I-8, there was a dose proportional increase inC_(max) for PEA. Average PEA Cmax values after I-8 dosing were 25.3,46.9, and 102 ng/mL following the 4, 8, and 16 mg/kg doses,respectively. Average dose normalized AUC_(last) values (13.5, 14.8, and16.8 hr*kg*ng/mL/mg) and bioavailability (21.7, 23.9, and 27.1%) werealso after similar the 4, 8, and 16 mg/kg 1-8 doses, respectively.

Following each dose of I-16, there was a dose proportional increase inC_(max) for PEA. Average PEA C_(max) values after I-16 dosing were 25.3,43.9, 68.3 ng/mL following the 5.2, 10.35, and 20.7 mg/kg doses,respectively. Average dose normalized AUC_(last) values (9.08, 10.6,11.2 hr*kg*ng/mL/mg) and bioavailability (14.6, 17.0, 18.0%) were alsosimilar after the 5.2, 10.35, and 20.7 mg/kg I-16 doses, respectively.

Preparation of Dosing Formulations

Pro-drugs were dosed so that a total dose of 2.5, 5, 10 mg/kg of PEA wasadministered. Each prodrug was formulated in a vehicle comprised of 10%Solutol HS15, 10% n-methyl pyrrolidone (NMP), 10% polyethylene glycol400 (PEG400) and 70% water. Formulations were prepared fresh on the dayof dosing.

Animal Dosing

Pharmacokinetics of PEA were evaluated in fasted male Sprague-Dawleyrats. Rats were housed one per cage. Each rat was fitted with a jugularvein cannula (JVC) for blood collection. Each study group was dosing intriplicate. Rats were fasted for a minimum of twelve hours prior todosing. Food was returned at four hours post dosing. Animals had freeaccess to water throughout the study. Blood samples (˜300 μL) werecollected from the rats via a JVC and placed into chilled polypropylenetubes containing sodium heparin as an anticoagulant, and 30 μL of 0.5 Mcitric acid. Samples were maintained chilled throughout processing.Blood samples were centrifuged at 4° C. and 3,000 g for 5 minutes.Plasma (˜150 μL) was then transferred to a chilled, labeledpolypropylene tube containing 15 μL of 10% formic acid, placed on dryice, and stored in a freezer maintained at −60° C. to −80° C. Bloodsampling times are shown in Table 18a.

TABLE 18a Study Design. Dose Dosing Blood Total (mg/kg Solution DosingSample Test Dosing Animals of pro- Conc. Volume Time Group # ArticleRoute n = drug)* (mg/mL) (mL/kg) Vehicle Points 1 I-8 PO 3 4 2 2 20%Pre-dose, 2 PO 3 8 2 4 (Solutol 5, 15, 3 PO 3 16 3 5.3 HS15:NMP 30 min,1:1) 10% 1, 2, PEG400; 4, 8 hours 70% H₂O 4 I-16 PO 3 5.2 2 2.6 20%Pre-dose, 5 PO 3 10.35 3 3.45 (Solutol 5, 15, 6 PO 3 20.7 3 6.9 HS15:NMP30 min, 1:1) 10% 1, 2, PEG400; 4, 8 hours 70% H₂O *All doses are basedon mg/kg of the pro-drugs, and deliver 10 mg/kg of active drug, PEA.

An LC-MS/MS method for the determination of PEA and PEA-prodrug isdescribed above (see e.g., Example 3).

Pharmacokinetic parameters were calculated from the time course of theplasma concentration. Maximum plasma concentration (C_(max)) and thetime to reach maximum plasma drug concentration (T_(max)) after oraldosing were observed from the data. Area under the time concentrationcurve (AUC) was calculated using the linear trapezoidal rule withcalculation to the last quantifiable data point, and with extrapolationto infinity if applicable. At least three quantifiable data points wererequired to determine the AUC. Mean residence time (MRT) was calculatedby dividing the area under the moment curve (AUMC) by the AUC.Bioavailability was determined by dividing the individualdose-normalized PO AUC_(last) values by the average IV AUC_(last) value(IV data from Example 5). Samples below the limit of quantitation weretreated as zero for pharmacokinetic data analysis.

Results

No adverse reactions were observed following the oral administration ofPEA pro-drugs in male Sprague-Dawley rats. Dosing solutions were notanalyzed by LC-MS/MS. Nominal dosing level was used in calculations.Concentrations are expressed as mg/mL of the free base.

Individual and average plasma concentrations for PEA and are shown inTable 18b through Table 18g. Data are expressed as ng/mL of the freedrug. Samples that were below the limit of quantitation were not used inthe calculation of averages. Plasma concentration versus time data areplotted in FIGS. 10A through 10L. Endogenous levels of PEA were found inthe majority of all the rats. Measured concentrations of PEA in plasmasamples were corrected by subtracting the concentration of PEA measuredin the pre-dose samples. Corrected values are reported in tables belowand were used to determine pharmacokinetic parameters. Corrected valuesthat were negative are reported as not determined (ND).

TABLE 18b Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-8 in20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 4 mg/kg in MaleSprague-Dawley Rats. Oral (4 mg/kg I-8 equals 2.5 mg/kg PEA) Rat # Time(hr) 446 447 448 Mean SD 0 (pre-dose) BLOQ BLOQ BLOQ ND ND 0.083 6.36BLOQ BLOQ ND ND 0.25 4.60  2.71 BLOQ  3.66 ND 0.50 9.80 16.9 14.3 13.73.59 1.0 18.1  31.3 26.4 25.3 6.67 2.0 6.19  6.38 10.4  7.66 2.38 4.0BLOQ BLOQ  4.57 ND ND 8.0 BLOQ BLOQ BLOQ ND ND Animal Weight  0.294  0.287   0.294   0.292  0.004 (kg) Volume Dosed 0.59  0.57  0.59  0.580.01 (mL) C_(max) (ng/mL) 18.1  31.3 26.4 25.3 6.67 t_(max) (hr) 1.0  1.0  1.0  1.0 0.0  t_(1/2)(hr) ND³ ND³ ND³ ND ND MRT_(last) (hr)  0.991  0.988  1.59  1.19  0.345 AUC_(last) (hr 22.1  33.6 45.3 33.7 11.6 ng/mL) AUC_(∞) (hr ND³ ND³ ND³ ND ND ng/mL) Dose-normalized Values¹AUC_(last) (hr kg 8.84 13.4 18.1 13.5 4.65 ng/mL/mg) AUC_(∞) (hr kg ND³ND³ ND³ ND ND ng/mL/mg) Bioavailability 14.2  21.6 29.2 21.7 7.48 (%)²C_(max): maximum plasma concentration; t_(max): time of maximum plasmaconcentration; t_(1/2): half-life, data points used for half-lifedetermination are in bold; MRT_(last): mean residence time, calculatedto the last observable time point; AUC_(last): area under the curve,calculated to the last observable time point; AUC_(∞): area under thecurve, extrapolated to infinity; ND: not determined; BLOQ: below thelimit of quantitation (2.5 ng/mL); ¹Dose-normalized by dividing theparameter by nominal dose in mg/kg; ²Bioavailability determined bydividing individual dose-normalized oral AUC_(last) values by theaverage IV AUC_(last) value 62.1 hr*ng/mL from Example 5; ³Notdetermined due to lack of quantifiable data points trailing the C_(max).

TABLE 18c Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-8 in20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 8 mg/kg in MaleSprague-Dawley Rats. Oral (8 mg/kg I-8 equals 5 mg/kg PEA) Rat # Time(hr) 449 450 451 Mean SD 0 (pre-dose) ND ND ND ND ND 0.083 3.16  1.410.920  1.83 1.18 0.25 9.62  1.31 2.86  4.60 4.42 0.50 46.2 30.0 13.730.0 16.3  1.0 61.8 43.7 35.1 46.9 13.7  2.0 9.52 18.4 22.0 16.6 6.414.0 0.0300  1.13 3.37  1.51 1.70 8.0 BLOQ BLOQ BLOQ ND ND Animal Weight(kg) 0.302   0.301 0.287   0.297  0.008 Volume Dosed (mL) 1.12  1.201.15  1.19 0.03 C_(max) (ng/mL) 61.8 43.7 35.1 46.9 13.6  t_(max) (hr)1.0  1.0 1.0  1.0 0.0  t_(1/2)(hr) ND³ ND³ ND³ ND ND MRT_(last) (hr⁾1.05  1.34 1.58  1.32  0.268 AUC_(last) (hr ng/mL) 80.4 73.2 68.5 74.05.97 AUC_(∞) (hr ng/mL) ND³ ND³ ND³ ND ND Dose-normalized Values¹AUC_(last) (hr kg 16.1 14.6 13.7 14.8 1.19 ng/mL/mg) AUC_(∞) (hr kg ND³ND³ ND³ ND ND ng/mL/mg) Bioavailability (%)² 25.9 23.6 22.1 23.9 1.92C_(max): maximum plasma concentration; t_(max): time of maximum plasmaconcentration; t_(1/2): half-life, data points used for half-lifedetermination are in bold; MRT_(last): mean residence time, calculatedto the last observable time point; AUC_(last): area under the curve,calculated to the last observable time point; AUC_(∞): area under thecurve, extrapolated to infinity; ND: not determined; BLOQ: below thelimit of quantitation (2.5 ng/mL); ¹Dose-normalized by dividing theparameter by nominal dose in mg/kg; ²Bioavailability determined bydividing individual dose-normalized oral AUC_(last) values by theaverage IV AUC_(last) value 62.1 hr*ng/mL from Example 5; ³Notdetermined due to lack of quantifiable data points trailing the C_(max).

TABLE 18d Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-8 in20% Solutol HS15:NMP (1:1), 10% PEG400, 70% H₂O) at 16 mg/kg in MaleSprague-Dawley Rats. Oral (16 mg/kg I-8 equals 5 mg/kg PEA) Rat # Time(hr) 452 453 454 Mean SD 0 (pre-dose) ND  ND  BLOQ ND ND 0.083 ND   2.605.10 3.85 ND 0.25 5.75 BLOQ 13.4 9.58 ND 0.50 70.2 45.0 80.6 65.3 18.3 1.0 95.8 74.4 137 102 31.8  2.0 28.6 38.0 46.0 37.5 8.72 4.0 6.97 13.36.56 8.94 3.78 8.0 BLOQ BLOQ BLOQ ND ND Animal Weight (kg) 0.288   0.2890.292 0.290  0.002 Volume Dosed (mL) 1.53  1.53 1.55 1.54 0.01 C_(max)(ng/mL) 95.8 74.4 137 102 31.8  t_(max) (hr) 1.0 1.0  1.0 1.0 0.0 t_(1/2)(hr) ND³ ND³ ND³ ND ND MRT_(last) (hr⁾ 1.33  1.62 1.33 1.43 0.162 AUC_(last) (hr ng/mL) 149 143   212 168 38.0  AUC_(∞) (hr ng/mL)ND³ ND³ ND³ ND ND Dose-normalized Values¹ AUC_(last) (hr kg 14.9 14.321.2 16.8 3.80 ng/mL/mg) AUC_(∞) (hr kg ND³ ND³ ND³ ND ND ng/mL/mg)Bioavailability (%)² 24.0 23.1 34.1 27.1 6.13 C_(max): maximum plasmaconcentration; t_(max): time of maximum plasma concentration; t_(1/2):half-life, data points used for half-life determination are in bold;MRT_(last): mean residence time, calculated to the last observable timepoint; AUC_(last): area under the curve, calculated to the lastobservable time point; AUC_(∞) : area under the curve, extrapolated toinfinity; ND: not determined; BLOQ: below the limit of quantitation (2.5ng/mL); ¹Dose-normalized by dividing the parameter by nominal dose inmg/kg; ²Bioavailability determined by dividing individualdose-normalized oral AUC_(last) values by the average IV AUC_(last)value 62.1 hr*ng/mL from Example 5; ³Not determined due to lack ofquantifiable data points trailing the C_(max).

TABLE 18e Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-16 in20% Solutol HS15:NMP (1:1). 10% PEG400, 70% H₂O) at 5.2 mg; kg in MaleSprague-Dawley Rats. Oral (5.2 mg/kg I-16 equals 2.5 mg/kg PEA) Rat #Time (hr) 455 456 457 Mean SD 0 (pre-dose) ND ND ND  ND ND 0.083 ND NDND  ND ND 0.25 ND ND  2.57 ND ND 0.50 2.83 6.02 52.2 20.3  27.6  1.015.1  8.52 25.5 16.4  8.53 2.0 5.93 3.51  6.25 5.23 1.50 4.0 ND ND ND ND ND 8.0 BLOQ BLOQ BLOQ ND ND Animal Weight (kg)  0.284  0.283   0.285 0.284  0.001 Volume Dosed (mL) 0.74 0.74  0.74 0.74 0.00 C_(max)(ng/mL) 15.1  8.52 52.2 25.3  23.6  t_(max) (hr) 1.0  1.0   0.50 0.830.29 t_(1/2)(hr)  ND³  ND³ ND³ ND ND MRT_(last) (hr) 1.16 1.06   0.8331.02  0.167 AUC_(last) (hr ng/mL) 15.4  10.4  42.4 22.7  17.2  AUC_(∞)(hr ng/mL)  ND³  ND³ ND³ ND ND Dose-normalized Values¹ AUC_(last) (hr kg6.14 4.16 16.9 9.08 6.88 ng/mL/mg) AUC_(∞) (hr kg  ND³  ND³ ND³ ND NDng/mL/mg) Bioavailability (%)² 9.89 6.70 27.3 14.6  11.1  C_(max):maximum plasma concentration; t_(max): time of maximum plasmaconcentration; t_(1/2): half-life, data points used for half-lifedetermination are in bold; MRT_(last): mean residence time, calculatedto the last observable time point; AUC_(last): area under the curve,calculated to the last observable time point; AUC_(∞): area under thecurve, extrapolated to infinity; ND: not determined; BLOQ: below thelimit of quantitation (2.5 ng/mL); ¹Dose-normalized by dividing theparameter by nominal dose in mg/kg; ²Bioavailability determined bydividing individual dose-normalized oral AUC_(last) values by theaverage IV AUC_(last) value 62.1 hr*ng/mL from Example 5; ³Notdetermined due to lack of quantifiable data points trailing the C_(max).

TABLE 18f Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-16 in20% Solutol HS15:NMP (1:1). 10% PEG400, 70% H₂O) at 10.35 mg/kg in MaleSprague-Dawley Rats. Oral (10.35 mg/kg I-16 equals 2.5 mg/kg PEA) Rat #Time (hr) 458 459 460 Mean SD 0 (pre-dose) ND  ND  ND  ND ND 0.083 ND ND  ND  ND ND 0.25 0.400 ND    0.960   0.680 ND 0.50 30.0 11.4 24.4 21.99.52 1.0 47.2 35.5 49.0 43.9 7.31 2.0 5.98 12.5 16.1 11.5 5.13 4.0 ND  5.02 ND  ND ND 8.0 BLOQ BLOQ BLOQ ND ND Animal Weight 0.288   0.298  0.284   0.290  0.007 (kg) Volume Dosed 0.99  1.03  0.98  1.00 0.03(mL) C_(max) (ng/mL) 47.2 35.5 49.0 43.9 7.33 t_(max) (hr) 1.0  1.0  1.0 1.0 0.0  t_(1/2)(hr) ND³ ND³ ND³ ND ND MRT_(last) (hr) 0.946  1.58 1.06  1.20  0.337 AUC_(last) (hr 49.7 54.7 54.2 52.8 2.72 ng/mL)AUC_(∞) (hr ND³ ND³ ND³ ND ND ng/mL) Dose-normalized Value¹ AUC_(last)(hr kg 9.94 10.9 10.8 10.6  0.544 ng/mL/mg) AUC_(∞) (hr kg ND³ ND³ ND³ND ND ng/mL/mg) Bioavailability 16.0 17.6 17.4 17.0  0.876 (%)² C_(max):maximum plasma concentration; t_(max): time of maximum plasmaconcentration; t_(1/2): half-life, data points used for half-lifedetermination are in bold; MRT_(last): mean residence time, calculatedto the last observable time point; AUC_(last): area under the curve,calculated to the last observable time point; AUC_(∞),: area under thecurve, extrapolated to infinity; ND: not determined; BLOQ: below thelimit of quantitation (2.5 ng/mL); ¹Dose-normalized by dividing theparameter by nominal dose in mg/kg; ²Bioavailability determined bydividing individual dose-normalized oral AUC_(last) values by theaverage IV AUC_(last) value 62.1 hr*ng/mL from Example 5; ³Notdetermined due to lack of quantifiable data points trailing the C_(max).

TABLE 18g Individual and Average Plasma Concentrations (ng/mL) andPharmacokinetic Parameters for PEA after Oral Administration of I-16 in20% Solutol HS15:NMP (1:1). 10% PEG400, 70% H₂O) at 20.7 mg/kg in MaleSprague-Dawley Rats. Oral (20.7 mg/kg I-16 equals 2.5 mg/kg PEA) Rat #Time (hr) 461 462 463 Mean SD 0 (pre-dose) ND  ND  ND  ND ND 0.083 ND 1.53 1.96 1.75 ND 0.25  4.19 10.8 9.44 8.14 3.49 0.50 31.1 30.7 30.830.9  0.225 1.0 64.0 81.2 59.6 68.3 11.4  2.0 33.4 20.5 24.5 26.2 6.624.0 12.6 2.68 8.74 8.02 5.02 8.0 ND  BLOQ BLOQ ND ND Animal Weight (kg)  0.280 0.284 0.285 0.283  0.003 Volume Dosed (mL)  1.93 1.96 1.97 1.950.02 C_(max) (ng/mL) 64.0 8.2 59.6 68.3 11.4  t_(max) (hr)  1.0 1.0 1.01.0 0.0  t_(1/2)(hr) ND³ ND³ ND³ ND ND MRT_(last) (hr)  1.66 1.29 1.531.49  0.190 AUC_(last) (hr ng/mL) 123   108 104 112 10.1  AUC_(∞) (hrng/mL) ND³ ND³ ND³ ND ND Dose-normalized Values¹ AUC_(last) (hr kg 12.310.8 10.4 11.2 1.01 ng/mL/mg) AUC_(∞) (hr kg ND³ ND³ ND³ ND ND ng/mL/mg)Bioavailability (%)² 19.8 17.4 16.7 18.0 1.63 C_(max): maximum plasmaconcentration; t_(max): time of maximum plasma concentration; t_(1/2):half-life, data points used for half-life determination are in bold;MRT_(last): mean residence time, calculated to the last observable timepoint; AUC_(last): area under the curve, calculated to the lastobservable time point; AUC_(∞): area under the curve, extrapolated toinfinity; ND: not determined; BLOQ: below the limit of quantitation (2.5ng/mL); ¹Dose-normalized by dividing the parameter by nominal dose inmg/kg; ²Bioavailability determined by dividing individualdose-normalized oral AUC_(last) values by the average IV AUC_(last)value 62.1 hr*ng/mL from Example 5; ³Not determined due to lack ofquantifiable data points trailing the C_(max).

Example 19: Synthesis of Compound I-9

A mixture of solketal (21 gm, 0.16 mol), succinic anhydride (15.9 gm,0.16 mol) and pyridine (500 mL) was heated to reflux for 16 hrs.Conversion was monitored by NMR. Pyridine was removed under high vacuum.Approximately 15-20% pyridine was still remaining. Mixture was taken tonext step as is without further purification.

A solution of INT-19a (62.13 mg, 0.27 mol) and PEA (61.46 mg, 0.27 mol)in DCM (1 lire) was cooled to 0° C. To this solution was added1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (152.5 mg, 0.8 mol)followed by 4-dimethylaminopyridine (DMAP) (9.8 mg, 0.08 mol) inportions. Reaction mixture was warmed to room temperature and stirredfor 24 hrs. Reaction mixture was washed with water and brine andextracted with DCM. Organic layers were separated and dried overanhydrous sodium sulfate, filtered and concentrated. Crude material waspurified by column chromatography with hexanes and ethyl acetate toobtain 88 mg of pure INT-19b as white solid.

INT-19b (88 mg) was dissolved in methanol (4 Liter) and cooled to 5° C.To this solution was added Dowex H* resin (45 mg) and stirred at 5° C.for 8 hrs. Then resin was filtered off with a pad of celite. Filtratewas concentrated to obtain off white solid. Solids were recrystallizedwith ethyl acetate to give 73.6 mg of pure I-9.

Example 20: General Synthesis of Diester PEA Prodrugs

Compounds of the present invention may be synthesized according toScheme 20.

A solution of PEA prodrug (1 eq), RCOOH (2.2-3.0 eq) in DCM (10 vol) wascooled to 0° C. To this solution was added1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (3 eq) followed byaddition of 4-dimethylaminopyridine (DMAP) (0.3 eq) in portions.Progress of the reaction was monitored by TLC/NMR. After conversion, thereaction mixture is diluted with DCM, washed with water, sat.aq sodiumbicarbonate and brine and extracted with DCM. The organic layers weredried over anhydrous sodium sulfate, filtered and concentrated. Thecrude residue was purified by column chromatography with increasinggradient of ethylacetate in hexanes.

Example 21: Synthesis of Compounds I-8 and I-16

Compounds I-8 and I-16 may be synthesized according to Schemes 21a and21h.

Example 22: Assessing the Analgesic Effects of I-16 in theCarrageenan-Induced Inflammatory Model Using Thermal HyperalgesiaTesting

Sixty male Sprague Dawley rats were used in this study. Baseline thermalhyperalgesia thresholds were determined on day -1; animals were dividedinto 6 groups based on baseline thermal hyperalgesia thresholds. On day0, animals received an oral dose of vehicle or I-16. Approximately 30minutes after dosing the animals received an intra-plantar injection of2% carrageenan solution. The animals were assessed for thermalhyperalgesia approximately 4 and approximately 24 hours aftercarrageenan injection.

Thermal hyperalgesia was assessed at baseline (prior to dosing witheither vehicle or I-16), 4 hours, and 24 hours post-carrageenaninjection. Oral administration of 10.25 mg/kg (equivalent to 5 mg/kgequivalents of PEA) I-16 did not significantly reduce the thermalhyperalgesia induced by carrageenan injection into the hind paw at anytime point. Oral administration of 20.50 mg/kg (equivalent to 15 mg/kgof PEA) I-16 significantly reduced thermal hyperalgesia at the 4-hourtime point, but did not significantly reduce thermal hyperalgesia at the24-hour time point (FIG. 11).

Mean±SEM ipsilateral paw withdrawal latencies following carrageenaninjection in vehicle and I-16 treated animals during the pharmacologicalassessment period (day 0). All animals received a mixture of 10%solutol, 10% n-methyl pyrrolidone, 10% PEG 400, and 70% water (10 mL/kg)or I-16 (10.25 or 20.50 mg/kg) via oral gavage (n=10).

These results indicate that administration of I-16 significantly reducesthe degree of thermal hyperalgesia associated with inflammatory pain.Administration of I-16 produced a dose- and time-dependent reduction ofthermal hyperalgesia with administration of 10.25 mg/kg I-16 producingno significant effect, and administration of 20.50 mg/kg I-16significantly reducing thermal hyperalgesia at the 4-hour time point.

Example 23: Evaluation of Analgesic Effects in Rat Chronic ConstrictionInjury (CCI) Model

Two test compounds (I-16 and Gabapentin) were formulated in 15% Solutol®HS15/15% Polyethylene glycol (PEG) 400/70% water for injection (WFI) fororal (PO) administrations for 17 consecutive days (qdx17). A dosingvolume of 10 mL/kg was applied.

Methods:

Male Sprague Dawley rats weighing 180±20 g were used. Underpentobarbital (50 mg/kg, 5 ml/kg, IP) anesthesia, the left sciatic nervewas exposed at mid-thigh level. Four chromic gut ligatures, about 1 mmapart, were loosely tied around the nerve. The animals were then housedsocially in cages with soft bedding for at least 10 days before testingfor mechanical allodynia and thermal hyperalgesia.

Mechanical Allodynia

The rats were placed under inverted Plexiglas cages on a wire mesh rackand allowed to acclimate for 20 to 30 minutes. Allodynia was evaluatedby the Chaplin up/down method using von Frey filaments to the plantarsurface of the left hind paw. All rats were assessed for mechanicalallodynia for pre-surgical allodynia threshold on Day -3 (pre-surgerybaseline). For gabapentin group, the rats were pre-selected forexperimentation only if the pain threshold on Day 13 after nerveligation (pre-treatment) is reduced by 10 grams of force relative to theresponse of the individual paw before nerve ligation (pre-ligation),namely, with clear presence of allodynia. On Day 14, the mechanicalallodynia test was performed at 1 hour after administrations of I-16,vehicle, or gabapentin.

Thermal Hyperalgesia

Thermal hyperalgesia was measured by the IITC Model-336G (IITC Inc.,USA) apparatus. Each rat was placed within a plastic box atop a glassfloor for 20 to 30 minutes. A light beam under the floor was aimed atthe plantar surface of the left hind paw. The time was measuredautomatically when the paw was withdrawn away from the thermal stimulus.A cut-off latency of 23 sec was imposed. The latency to withdrawal isobtained for each rat and defined as the heat pain threshold. All ratswere assessed for thermal hyperalgsia for pre-surgical threshold on Day−3 (pre-surgery baseline). For gabapentin group, the rats werepre-selected for experimentation only if the pain threshold on Day 13after nerve ligation (pre-treatment) is reduced by 15 seconds. On Day14, the thermal hyperalgesia test was performed at 1.5 hour afteradministrations of I-16, vehicle, or gabapentin.

Group differences were compared to the vehicle control group.Differences were considered significant at P<0.05.

The formulations and dosing protocols are provided in Table 23 andvisualized in FIG. 12A:

TABLE 23 Test Conc. Dosage Dosage Rats Group Article Route mg/mL mL/kgmg/kg (Male) 1 Vehicle^(a) PO NA 10 NA, qd × 17 8^(b) (Days −2~14) 2Gaba- PO 10 10 100, qd × 1 8^(c) pentin 3 I-16 PO 3.1 10 31, qd × 178^(b) (Days −2~14) ^(a)Vehicle: 15% solutol ® HS15, 15% PEG400 & 70%WFI. Dose preparation instructions: first mix the TA in solutol, thenadd PEG400 and vortex, then add water and vortex to ensure a clearsolution. ^(b)The rats were randomized on Day −3. ^(c)The rats wererandomized on Day 13. Each group was underwent mechanical allodynia andthermal hyperalgesia testing on Day 14.

The results of an assay measuring the mechanical allodynia at posttreatment (1-hr) and (1.5-hr) is reported in FIG. 12B.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

1. A compound which is:

or a salt thereof.
 2. The compound of claim 1, wherein the compound is asalt.
 3. The compound of claim 2, wherein the salt is a pharmaceuticallyacceptable salt.
 4. A pharmaceutical composition comprising the compoundof claim 1 or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable excipient.
 5. The pharmaceutical compositionof claim 4, comprising one or more additional therapeutic agents.
 6. Thepharmaceutical composition of claim 4, which is formulated for oraldelivery.
 7. The pharmaceutical composition of claim 6, which isformulated as a solid formulation.
 8. The pharmaceutical compositionaccording to claim 7, wherein the solid formulation is a capsule.
 9. Thepharmaceutical composition according to claim 8, wherein the capsuleencloses a liquid.
 10. A method of treating pain comprisingadministering the compound according to claim 1 or a pharmaceuticallyacceptable salt thereof to a patient in need thereof.
 11. A method oftreating inflammatory pain comprising administering the compoundaccording to claim 1 or a pharmaceutically acceptable salt thereof to apatient in need thereof.
 12. A method of treating neuropathic paincomprising administering the compound according to claim 1 or apharmaceutically acceptable salt thereof to a patient in need thereof.13. A method of treating pain comprising administering thepharmaceutical composition according to claim 4 to a patient in needthereof.
 14. A method of treating inflammatory pain comprisingadministering the pharmaceutical composition according to claim 4 to apatient in need thereof.
 15. A method of treating neuropathic paincomprising administering the pharmaceutical composition according toclaim 4 to a patient in need thereof.